Methods of screening agents for activity using teleosts

ABSTRACT

The present invention provides methods of screening an agent for activity using teleosts. Methods of screening an agent for angiogenesis activity, toxic activity and an effect cell death activity in teleosts are provided. The invention further provides high throughput methods of screening agents in multi-well plates.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. Ser. No. 10/678,765,filed Oct. 2, 2003, which is a continuation of U.S. Ser. No. 09/645,432,filed Aug. 23, 2000, now U.S. Pat. No. 6,656,449, which is acontinuation-in-part of U.S. Ser. No. 09/255,397, filed Feb. 22, 1999,now U.S. Pat. No. 6,299,858, which derives priority from U.S.Provisional Patent Application Ser. No. 60/075,783, filed on Feb. 23,1998, and U.S. Provisional Patent Application Ser. 60/100,950, filed onSep. 18, 1998, each of which is incorporated herein by reference in itsentirety for all purposes. Commonly owned copending U.S. PatentApplication Ser. No. 60/110,464, filed Dec. 1, 1998 is directed torelated subject matter and is incorporated herein by reference in itsentirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was supported by a grant from the National Institutes ofHealth (Grant No. 1R43CA7938-01). The Government may have certain rightsin this invention.

BACKGROUND OF THE INVENTION

Currently, searches for target-specific therapeutic and prophylacticcompounds that have the ability to enhance or inhibit angiogenesisactivity, enhance or inhibit cell death activity, and/or exhibit lowtoxicity comprise three major focuses of drug discovery and development.Angiogenesis plays an important role not only in the further developmentof the embryonic vasculature, but also in many post-natal processes,such as wound healing and tissue and organ regeneration. Angiogenesishas also been identified as a critical process for solid tumor growth.Furthermore, uncontrolled blood cell proliferation and excessiveangiogenesis have been shown to constitute significant pathogeniccomponents in numerous diseases, including rheumatoid arthritis,atherosclerosis, diabetes mellitus, retinopathies, psoriasis, andretrolental fibroplasia.

Methods of screening agents for an ability to inhibit or enhanceangiogenesis activity would be useful in identifying those agents thatwould be effective in therapeutic or prophylactic treatment of a varietyof diseases involving angiogenesis processes. For example, angiogenesisinhibition is a powerful potential approach for ameliorating cancer(Folkman, New Eng. J. Med. 333:1757-1763 (1995); Kerbel, Nature 390:355(1997)) and for reversing blood vessel loss associated with tissueischemia, such as diabetic retinopathy (Bonn, Lancet 348:604 (1996);Breier et al, Haemist. 78(1):678-683 (1997). It appears thatanti-angiogenic therapies do not induce acquired drug resistance (Boehmet al., Nature 390:404-407 (1997))—a major problem with current cancertherapies. However, few therapeutic candidate molecules exist. It wouldtherefore be desirable to provide methods of identifying compounds thatinhibit angiogenesis and have therapeutic activities against diseasesthat would benefit from angiogenesis inhibition, such as cancer anddiabetes. Similarly, methods of screening for compounds that enhanceangiogenesis by stimulating blood vessel formation would be advantageousfor use in minimally invasive approaches for improving circulatoryfunction in various diseases, such as coronary artery disease,congestive heart failure, peripheral arterial disease, and peripheralvenous disease. Unfortunately, many current assays for angiogenesis donot permit in vivo assessment of compounds or their side effects inwhole animal models, or in multiple tissues or organs of animal modelssimultaneously and over time. In addition, many current assays forangiogenesis activity are not suitable for rapid, automated, orextensive compound screening, particularly screening of compoundlibraries containing numerous compounds, due to their complexity.

The search for compounds that can regulate promote or inhibit cell deathhas also been of vital interest. Necrosis and apoptosis are two knowntypes of cell death. Necrosis involves the pathologic death of livingtissue in a subject due to non-physiological injury to cells of thetissue. Apoptosis, which involves programmed cell death, is aphysiological process that ensures that an equilibrium is maintainedbetween cell proliferation and cell differentiation in mostself-renewing tissues of multicellular organisms. Regulation of celldeath activity (in particular, apoptosis) constitutes an importantmechanism in therapeutic and prophylactic approaches to many diseases,including, e.g., cancer and organ transplantation. Existing models forassessing apoptosis activity include the nematode worm, C. elegans.Although the nematode has many advantages as a model system, it is notthe optimum model for evaluating vertebrate cell death activity or foruse in screening compounds for potential therapeutic activity invertebrates.

There are currently two approaches for detecting cell death activity invertebrate hosts. The first approach uses standard cell culturetechniques and typically relies on standard microplate readers to detectthe death of cells cultured from an organism. A major drawback of thecell culture assay format is that it does not permit analysis of theeffects of a compound on cell types that have not been cultured (i.e.,other cell types). It also does not allow evaluation of the effects of acompounds on specific tissues or organs or in an intact whole host invivo. Furthermore, such an assay format does not permit the monitoringof cell death activities in multiple tissues, organs, or systems of alive host simultaneously or the continued monitoring of such activitiesover time. In addition, the cell culture assay approach does not allowfor rapid or automated high-throughput screening of many compounds.

A second approach to detecting cell death activity utilizes ahistochemical staining technique, designated terminal deoxyuridinenucleotide end labeling (TUNEL), to detect dead or dying cells insectioned tissues of vertebrate embryos. Unfortunately, with thisapproach, only a single time point in the life cycle of the host can beexamined; the death of cells in various tissues or organs of the subjectover a period of time cannot be monitored. Because many degenerativediseases occur in stages, the ability to examine changes in the patternof cell death activity caused by a compound and the duration of directand side effects of the compound on multiple tissues and organs wouldrepresent a significant improvement over such methods. Moreover, becausethe TUNEL approach requires that cells be fixed for visualization,effects in a live animal cannot be monitored.

The identification of target-specific therapeutic and prophylacticcompounds that exhibit low toxicity and/or side effects has also beenfocal point of drug discovery and development. Evaluation of thepotential impact of different compounds on humans and animal health is amajor component of risk assessment. There is increasing concern thatcurrent toxicity test procedures are inadequate. A number of cell-basedin vitro toxicity screens have been developed. Significantly, however,these screens do not permit evaluation of the toxic effects of acompound in vivo on an intact animal. Notably, these cell-based assaysare designed at the molecular and cellular levels; as a result,determining the impact of a compound of interest on higher levels ofcellular organization, such as the circulatory system andneurodevelopment, still requires subsequent whole animal testing. Inaddition, current screens do not permit the assessment of drug effectson all potential target cells, tissues, or organs of an animal. Nor canthe effects of a compound on multiple target tissues and organs bestudied simultaneously or over time using current assays. Underscoringthe need for the development of more predictive and comprehensivetoxicity screening methods, many compounds that pass preliminarycell-based testing fail final large animal toxicity testing, aprerequisite for eventual FDA approval. Furthermore, some potentialtherapeutic compounds that do not produce immediate lethality inducetoxic effects in specific organs and tissues. There is a need for acost-effective, comprehensive methods for screening compounds for toxicactivity in whole animals and in one or more designated target tissuesand organs in vivo and in cells in vitro and over time.

SUMMARY OF THE INVENTION

The present invention relates generally to methods of screening an agentfor an activity in a teleost. In one aspect, methods of screening anagent for an angiogenesis activity in vivo or in vitro are provided.Some such methods comprise administering the agent to a whole teleost invivo and detecting a response in the teleost or in at least one tissueor organ of the teleost indicating the angiogenesis activity. Other suchmethods comprise administering the agent to cells of a teleost in vitroand detecting a response in such cells indicating the angiogenesisactivity. In some such methods, the response is a reduction in bloodvessel formation relative to an untreated teleost. In other suchmethods, the response is an increase in blood vessel formation relativeto an untreated teleost.

In another aspect, the invention provides methods of screening an agentfor an effect on cell death activity in vivo or in vitro. Some suchmethods comprise administering the agent to a whole teleost in vivo anddetecting a response in the teleost or in at least one tissue or organof the teleost or cells thereof indicating an effect on cell deathactivity. Some such methods comprise administering the agent to cells ofa teleost in vitro and detecting a response in such cell indicating aneffect on cell death activity. In some such methods, the response is anincrease in cell death activity. In other such methods, the response isa decrease in cell death activity. The cell death activity may compriseapoptotic or necrotic activity. In some such methods, a fluorescent dyewhich labels dead or dying cells is administered to facilitate detectionof cell death activity. In some such methods, the fluorescent dye isadministered to the teleost prior to the administration of the agent. Insome such methods, the fluorescent dye is an unsymmetrical cyanine dye,such as a quinolium dye.

Also provided are methods of screening an agent for toxic activity invivo or in vitro. Some such methods comprise administering the agent toa whole teleost in vivo and detecting a response in the teleost or in atleast one tissue or organ of the teleost indicating toxicity. Other suchmethods comprise administering the agent in vitro to cells of a teleostand detecting a response in the cells indicating toxicity. In some suchmethods, the response is detected in two or more organs of the teleostsimultaneously.

In another aspect, the present invention provides methods of screeningan agent for angiogenesis activity and toxicity in vivo or in vitro.Some such methods comprise administering the agent to a whole teleost invivo and detecting a response in the teleost indicating angiogenesisactivity and/or toxicity. Other such methods comprise administering theagent in vitro to cells of a teleost and detecting a response in thecells indicating angiogenesis activity and/or toxicity.

In yet another aspect, the present invention includes methods ofscreening an agent for angiogenesis activity and an effect on cell deathactivity in vivo or in vitro. Some such methods comprise administeringthe agent to a teleost in vivo and detecting a response in the teleostindicating angiogenesis activity and/or an effect on cell deathactivity. Other such methods comprise administering the agent in vitroto cells of a teleost and detecting a response in the cells indicatingangiogenesis activity and/or an effect on cell death activity.

The present invention also includes methods of screening an agent for aneffect on cell death activity and toxic activity in vitro or in vivo.Some such methods comprise administering the agent in vivo to a teleostand detecting a response in the teleost indicating an effect on celldeath activity and/or toxicity. Other such methods compriseadministering the agent in vitro to cells of a teleost and detecting aresponse in the cells indicating an effect on cell death activity and/ortoxicity.

The invention further provides methods of screening an agent for apharmacological activity. Such methods entail providing a multi-wellplate, the wells containing teleosts. Agents are then into differentwells of the multi-well plate, and incubating the agents with theteleosts for sufficient time to induce a response in the teleostsindicative of the pharmacological activity. A labelling reagent isintroduced into each well, which, through processing by or binding to acomponent of the teleost, generates a detectable signal dependent on theextent of the response in the teleost. A signal is detected in each wellas an indication of the pharmacological activity of the agent introducedin the well. In some methods, the detectable signal is an opticallydetectable signal, which can be detected, for example, by a microplatereader. In some methods, a single teleost occupies in each well. In somemethods, the teleosts are zebrafish. In some methods, the teleosts aresynchronized embryos.

In some methods, the labelling reagent is a substrate of an enzyme, andthe response is an increase or decrease in activity of the enzyme. Insome methods, the labelling reagent comprises an antibody, and thedetectable signal is generated by the antibody bound to a cellularreceptor of the teleost. In some methods, the response is a change innumber of cells or types of cells of the teleost. In some methods, thelabelling reagent is a nucleic acid, and the detectable signal isgenerated by the nucleic acid bound to a nucleic acid of the teleost. Insome methods, the labelling reagent is contacted with a second labellingreagent that binds to the labelling reagent thereby generating thedetectable signal. In some methods, the pharmacological activity ismodulation of angiogenesis, the response is a change in alkalinephosphatase activity of the teleost and the labelling reagent is asubstrate for alkaline phosphatase. In some methods, the pharmacologicalactivity is modulation of apoptosis, the response is a change in levelof a caspase activity of the teleost and the labelling reagent is asubstrate for the caspase.

In some methods, optical density is determined within each well todistinguish teleosts that survive incubation with the agent and teleoststhat die due to incubation with the agent. In some methods, subsequentsteps of introducing a labelling agent and detecting signal areperformed on a subset of wells containing teleosts that surviveincubation with the compound. Some methods are followed by performing aconfirmatory assay on a subset of agents indicated to have thepharmacological activity. The confirmatory assay entails detecting asecond response of the teleosts, the same or different than theresponse, wherein the second response is a confirmatory indicator of thepharmacological activity. In some such methods, the response and thesecond response are the same, and the response is detected withpreformed with a plate reader, and the second response is detected witha microscope. Some methods further comprise determining an LD50 on asubset of agents indicated to have pharmacological activity. Somemethods further comprise determining organ-specific toxicity on a subsetof agents indicated to have pharmacological activity.

The invention further provides methods of monitoring distribution of anagent between teleosts and a surrounding medium. In such methods, amulti-well plate is provided in which the wells containing teleosts in amedium, and either the medium or the teleosts or both containing anagent. The teleosts are cultured in the wells. In each well, the amountof the agent in the medium, in the zebrafish or both is determined. Suchmethods can be used to determine absorption rate, excretion rate ofmetabolism rate of the agent.

The invention further provides methods of screening an agent for aproperty. Such methods entail providing a multi-well plate, the wellscontaining teleosts. Agents are introduced into different wells of themulti-well plate, and the agents are incubated with the teleosts forsufficient time to induce a response. The response is detected in eachwell as an indication of the property of the agent introduced in thewell.

A further understanding of the nature and advantages of the inventionsherein may be realized by reference to the detailed description of thespecification and the associated figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic diagram showing the processes of vasculargenesisand angiogenesis.

FIGS. 2A, 2B, and 2C are photographs through a dissecting microscopeshowing lateral views of zebrafish embryos at 72 hours (hr) ofdevelopment. The embryos have been stained with alkaline phosphatase(AP). Blood vessels are visualized by light microscopy after alkalinephosphatase staining. A control embryo (FIG. 2A) treated with 0.1%dimethyl sulfoxide (DMSO) has normal morphology and vessel formation.The subintestinal vessels (SIVs) (arrow) are in the characteristicpattern. An embryo treated with a fumagillin derivative at concentrationof 10 micromolar (μM) (FIG. 2B) shows both developmental delay (reducedfin size and axial length) and loss of the SIVs (arrow). The pronephericduct provides a positive control for AP staining (arrowhead). An embryotreated with a fumagillin derivative at a concentration of 100 μM (FIG.2C) is dead. Fumagillin derivatives induce developmental delay and toxicresponse in the embryos. The eye (E), yolk (Y) and fin (F) of theembryos are labeled for orientation. Scale bar=100 μm.

FIGS. 3A and 3B are photographs through a dissecting microscope showingtwo lateral views of zebrafish embryos at 72 hours of development. Eachembryo has been stained with alkaline phosphatase. FIG. 3A represents acontrol; FIG. 3B shows a treated embryos treated with a compound fromthe NCI library. Both embryos are morphologically normal, however, thetreated embryo has failed to form any SIVs (arrow) and shows a specificloss of the subintestinal vessels. The eye (E), yolk (Y) and fin (F) ofthe embryos are labeled for orientation. Scale bar=100 μm.

FIG. 4 is a photograph through a dissecting microscope of a lateral viewof an alkaline-phosphatase stained zebrafish embryo at 72 hours ofdevelopment. The embryo has been treated with a compound which inducedtruncation, pericardial edema (arrow), and reduction of SIV formation(arrowhead). A loss of lateral vessels in the SIV basket is shown. Theeye (E), yolk (Y) and fin (F) of the embryo are labeled for orientation.Scale bar=100 μm.

FIG. 5 is a photograph through a dissecting microscope showing a lateralview of a zebrafish embryo treated with a compound which inducedblebbing of the notocord (arrow), but did not effect SIV formation(arrowhead). The embryo been stained with AP. Axial defects do notusually effect angiogenesis. The eye (E), yolk (Y) and fin (F) of theembryo are labeled for orientation. Scale bar=100 μm.

FIGS. 6A-6D are photographs through a dissecting microscope showinglateral views of zebrafish embryos at 72 hours of development. Embryoshave been stained with AP. When VEGF was injected into the yolk of anembryo (FIGS. 6A and 6C), two angiogenic phenotypes were observed: 1)the appearance of long spikes projecting from the subintestinal vesselbasket (long arrows); and 2) increased vessel diameters in thesubintestinal basket (arrowheads). When VEGF was injected into theperivitelline space (FIG. 6D) of an embryo, we observed fusion of largevessels, inappropriate vessel formation (arrow), as well as heart (longarrow) and developmental defects. Control embryos (FIG. 6B), in whichbuffer was injected into either the yolk of perivitelline space, werenormal. The eye (E), yolk (Y) and fin (F) of the embryos are labeled fororientation. Scale bar=100 μm.

FIG. 7 is a photograph through a compound microscope (10× objective) ofan alkaline phosphatase staining of zebrafish embryos at day three ofdevelopment. These dorsal views of an untreated (top) and a treated(bottom) embryo show the effect of the anti-angiogenesis drug,Ovicillin, on the subintestinal veins (arrows). In addition to causing areduction in the subintestinal vessels, the drug had other effects,including causing pericardial edema (arrowheads). In this figure, theletter “E” denotes the eye, and the letter “Y” denotes the yolkball.

FIG. 8 is a photograph through a compound microscope (20× objective)showing a wholemount RNA in situ hybridization with flk-1 performed on aday one zebrafish embryo. In this lateral view of the trunk,intersomitic vessels (arrows), which are sprouting from the dorsal aorta(A), are labeled with the probe. Anterior is to the left and dorsal isup.

FIG. 9 is a microangiograph showing lateral profile of a zebrafishembryo at day three of development, depicting the normal vascularpattern, including the cranial (C), intersegmental (I) and subintestinal(S) vessels. The letter “H” denotes the heart, and the letter “E”denotes the eye. The data to construct the microangiograph was acquiredfrom an epifluorescence microscope and processed using digital imageprocessing software.

FIGS. 10A and 10B are compound microscope photographs (10× objective)under a compound microscope showing, respectively, a phase image of anormal (FIG. 10A) zebrafish embryo and a retinoic acid-treated (FIG.10B) zebrafish embryo. The embryo treated with retinoic acid (vitamin Aacid, Sigma Chemical Co.) was exposed to 1 μM retinoic acid (RA) at 12to 14 hours. Apoptosis occurred in the hindbrain of the RA-treatedembryo, as evidenced by the disorganization of the hindbrain and thesignificant reduction in distance between the otic vesicle and the eyein the retinoic acid-treated embryo, as compared with the normal embryo(compare arrows in FIGS. 10A and 10B). The letter “E” denotes the eye,the letters “YB” signify the yolkball, and the letters “OT” denote theotic vesicle.

FIG. 11 is a photograph through a dissecting microscope showing alateral view of a 5 day zebrafish embryo stained withstreptavidin-conjugated peroxidase. Both the liver (arrows) and the gut(G) are stained. The eye (E) and the otic vesicle (OV) of the embryo arelabeled for orientation. The magnification is comparable to FIGS. 12Aand 12B.

FIGS. 12A and 12B are photographs through a dissecting microscope whichshow a dose response of zebrafish embryos to which specific dosages ofdexamethasone had been administered. Zebrafish embryos were treated forfive days with dexamethasone to determine the effect of dexamethasone onliver development and function. The embryos were fixed withparaformaldehyde and incubated with streptavidin-peroxidase to detectthe liver after incubating with a chromogenic dye. The arrows indicatethe position of the liver. FIG. 12A (top), six day old untreated embryo(control embryo); FIG. 12B (bottom), six day old embryo treated with 100μM of dexamethasone for five days. Embryos treated with dexamethasoneshowed a dramatic reduction in liver size compared with control embryos.Scale bar=1 millimeter (mm); Eye (E); Gut (G); Tail (T).

FIG. 13 is a graphical illustration showing a dose response of zebrafishembryos to various dosages of dexamethasone. Zebrafish embryos wereexposed to different concentrations of dexamethasone (i.e.,concentrations ranging from 1 μM to 100 μM) for five days as describedfor FIGS. 12A and 12B. After treatment, the embryos were stained with asoluble dye to detect liver defects specifically. After staining, theproduct formed by peroxidase was detected by absorbance at 405nanometers (nm). The values were expressed as a percentage of control (%Control), where the control (i.e., untreated embryos) is 100%. Thestandard deviation was also calculated and added to the data. Livertoxicity resulting from dexamethasone is suggested by a reduction inliver size.

FIG. 14 is a photograph of a six day old zebrafish embryo fixed withparaformaldehyde and stained for alkaline phosphatase. The arrowindicates the position of the stained embryonic kidney. Scale bar=1 mm;Eye (E); Gut (G); Tail (T).

FIG. 15: Flow chart of an exemplary high throughput screening assay.

FIG. 16A. Linear relationship between OD (405 nm) and pNPP incubationtime. Using 1 embryo/well at 24 (red), 48 (blue) and 72 (green) hpfstage, a linear relationship was observed between 20 and 40 minutes(each point represents mean+S.E., n=6).

FIG. 16B. OD (405 nm) difference among embryos aged 24, 48 and 72 hpf,incubated with pNPP for 5 (red), 10 (blue), 20 (green), 30 (dark green)and 40 (magenta) minutes (each point represents mean+S.E., n=6).

FIG. 17. Representative embryos re-stained for microscopic observationafter OD (405 nm) measurement. A. 24 hpf; B. 48 hpf; and C. 72 hpf. Thearrows indicate the site of the SIVs formation. At 24 and 48 hpf theSIVs are not yet present. In contrast, the SIVs are quite obvious by 72hpf.

FIG. 18. Dose response curve of SU5416 (each point represents mean+S.E.,n=10).

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which this invention belongs. The following referencesprovide one of skill with a general definition of many of the terms usedin this invention: Singleton et al., DICTIONARY OF MICROBIOLOGY ANDMOLECULAR BIOLOGY (2d ed. 1994); THE CAMBRIDGE DICTIONARY OF SCIENCE ANDTECHNOLOGY (Walker ed., 1988); and Hale & Marham, THE HARPER COLLINSDICTIONARY OF BIOLOGY (1991). As used herein, the following terms andphrases have the meanings ascribed to them unless specified otherwise.Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are described. Forpurposes of the present invention, the following terms and phrases areintended to have the following general meanings as they are used herein:

The term “subject” as used herein includes an animal. The term “animal”as used herein includes a vertebrate animal, such as a vertebrate fish.Vertebrate fish include teleosts, such as, e.g., zebrafish, medaka,Giant rerio, and puffer fish. The term “teleost” as used herein means ofor belonging to the Teleostei or Teleostomi, a group consisting ofnumerous fishes having bony skeletons and rayed fins. Teleosts include,for example, zebrafish, medaka, Giant rerio, and puffer fish.

The term “larva” or “larval” as used herein means the stage of any ofvarious animals, including vertebrate animals, such as vertebrate fishes(including teleosts, such as, e.g., zebrafish, medaka, Giant rerio, andpuffer fish), between embryogenesis and adult.

“Angiogenesis activity” or “angiogenic activity” in reference to anagent is defined herein as the ability of the agent to enhance, inhibit,or prevent the formation or outgrowth of blood vessels or lymph vessels.Angiogenesis activity or angiogenic activity in reference to a subjectrefers to activity associated with angiogenesis within a subject ororgan(s) or tissue(s) of a subject or originating from within a subjector organ(s) or tissues(s) of the subject.

“Anti-angiogenesis activity” or “anti-angiogenic activity” in referenceto an agent is defined herein as the ability of the agent to inhibit,prevent, or greatly reduce the formation or outgrowth of blood or lymphvessels, or destroy such vessels during sprouting or outgrowth.Anti-angiogenesis activity or anti-angiogenic activity in reference to asubject refers to activity associated with anti-angiogenesis within asubject or organ(s) or tissue(s) of a subject or originating from withina subject or organ(s) or tissues(s) of the subject.

The term “apoptotic activity” or “apoptosis activity” in reference to anagent is defined herein as the ability of the agent to enhance, inhibit,or prevent apoptosis. Apoptotic activity or apoptosis activity inreference to a subject refers to activity associated with the death ofcells within a subject or organ(s) or tissue(s) of a subject ororiginating from within a subject or organ(s) or tissues(s) of thesubject.

“Cell death activity” in reference to an agent is defined herein as theability of the agent to enhance, inhibit, or prevent the death of one ormore cells within a subject or organ(s) or tissue(s) of a subject ororiginating from within a subject or organ(s) or tissues(s) of thesubject. Cell death activity in reference to a subject refers toactivity associated with the death of cells within a subject or organ(s)or tissue(s) of a subject or originating from within a subject ororgan(s) or tissues(s) of the subject.

The term “necrotic activity” or “necrosis activity” in reference to anagent is defined herein as the ability of the agent to enhance, inhibit,or prevent necrosis.

An “effect on cell death activity” as used herein refers to the way inwhich an agent acts upon or influences cell death activity in a subject.Such effects include an ability to enhance or inhibit cell deathactivity in the subject, as indicated or manifested by, for example, aclinical manifestation, characteristic, symptom, or event that occurs oris observed in, associated with, or peculiar to death of cells in asubject.

An “effect on apoptotic activity” as used herein refers to the way inwhich an agent acts upon or influences apoptotic activity in a subject.Such effects include an ability to enhance or inhibit apoptotic activityin the subject, as indicated or manifested by, for example, a clinicalmanifestation, characteristic, symptom, or event that occurs or isobserved in, associated with, or peculiar to apoptosis of cells in asubject.

An “endogenously occurring” as used herein means occurring originatingfrom within.

The term “gene” is used broadly to refer to any segment of DNAassociated with a biological function. Thus, genes include codingsequences and/or the regulatory sequences required for their expression.Genes also include non-expressed DNA segments that, for example, formrecognition sequences for other proteins.

The term “nucleic acid” or “nucleic acid segment” refers to adeoxyribonucleotide or ribonucleotide and polymer thereof which is ineither single- or double-stranded form. Unless specifically limited, theterm encompasses nucleic acids containing known analogues (synthetic andnaturally occurring) of nucleotides, which have similar bindingproperties as the reference nucleic acid and are metabolized in a mannersimilar to the reference nucleotides. Unless otherwise indicated, aparticular nucleic acid sequence also implicitly encompassesconservatively modified variants thereof (e.g., degenerate codonsubstitutions) and complementary sequences, as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions canbe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991);Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al.,Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is usedinterchangeably with gene, cDNA, and mRNA encoded by a gene.

The term “isolated nucleic acid” or “isolated nucleic acid segment”means a single- or double-stranded nucleic acid (e.g., an RNA, DNA, or amixed polymer), which is substantially separated from other genome DNAsequences as well as proteins or complexes such as ribosomes andpolymerases, which naturally accompany a native sequence. The termembraces a nucleic acid sequence which has been removed from itsnaturally occurring environment, and includes recombinant or cloned DNAisolates and chemically synthesized analogues or analogues biologicallysynthesized by heterologous systems. A substantially pure moleculeincludes isolated forms of the molecule. An “isolated polypeptide” orprotein carries a similar meaning with the polypeptide or protein beingsubstantially separated from any cellular contaminants and componentsnaturally associated with the protein in vivo.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

A “chimeric molecule” as used herein refers to a linked moleculeobtained after conjugation of two or more different types of molecules(e.g., lipids, glycolipids, peptides, proteins, glycoproteins,carbohydrates, nucleic acids, natural products, synthetic compounds,organic molecule, inorganic molecule, etc.).

The term “normal blood vessel formation” as used herein refers to thetypical, usual, or natural process of forming or producing blood vesselsin a subject.

The term “gene expression profile” or “gene expression pattern” as usedherein means a profile or pattern based on the detection of mRNA foreach gene to be included in the profile or pattern. mRNA can be detectedat a particular time or under a particular condition(s). mRNA isextracted from cells, tissues, organs, or an entire organism of interestand detected. The amount or level of mRNA for a particular gene can bedetermined quantitatively.

The term “protein expression profile” or “protein expression pattern” asused herein means a profile or pattern based on the detection of aprotein. The protein can be detected at a particular time or under aparticular condition(s). Protein is extracted from cells, tissues,organs, or an entire organism of interest and detected. The amount orlevel of protein can be determined quantitatively.

The term “agent” includes any element, compound, or entity, including,but not limited to, e.g., pharmaceutical, therapeutic, pharmacologic,environmental or agricultural pollutant or compound, aquatic pollutant,cosmeceutical, drug, toxin, natural product, synthetic compound, orchemical compound.

The term “natural compound” as used herein includes a molecule isolated,extracted, or purified from a plant, animal, yeast, bacterium, or othermicroorganism. A natural compound includes, e.g., among other things,organic molecules belonging to the broad biochemical classes ofpeptides, proteins, glycoproteins, nucleic acids, carbohydrates, lipids,fats, glycolipids, as well as more complex molecules which comprise,e.g., elements of more than one of these basic biochemical classes.

The term “synthetic compound” as used herein includes a moleculesynthesized de novo or produced by modifying or derivatizing a naturalcompound.

“Developmental defect” as used herein means a deficiency, imperfection,or difference in the development of a tissue, organ, or other bodilycomponent of an animal relative to normal development. Such a defect isidentified as a change, difference, or lack of something necessary ordesirable for completion or proper operation in the development of atissue, organ, or other bodily component of the animal as compared withnormal development of the component. Developmental defects include, forexample, the failure of organ to develop properly, excess or reducedcell proliferation as compared to normal cell proliferation, and themalfunctioning of an organ or tissue.

Generally, the nomenclature used hereafter and the laboratory proceduresin cell culture, molecular genetics, and nucleic acid chemistrydescribed below are those well known and commonly employed in the art.Standard techniques such as described in Sambrook et al., MOLECULARCLONING: A LABORATORY MANUAL (Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 2nd ed. 1989) and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,Vols. 1-3 (Virginia Benson Chanda ed., John Wiley & Sons, 1994-1998),each of which is incorporated herein by reference in its entirety forall purposes, are used for recombinant nucleic acid methods, nucleicacid synthesis, cell culture, and transgene incorporation, e.g.,electroporation, injection, ingestion, and lipofection. Electroporationtechniques utilize a pulse of high electrical current to introducemolecules of interest into cells, tissues, or organisms. Lipofectionemploys lipid-like cationic molecules that interact strongly with cellmembranes, destabilizing them locally, thereby allowing DNA and RNAentry into cells. Generally, oligonucleotide synthesis and purificationsteps are performed according to the specifications. The techniques andprocedures are generally performed according to conventional methods inthe art and various general references which are provided throughoutthis document. The procedures therein are believed to be well known inthe art and are provided for the convenience of the reader.

The term “transgenic” in reference to an organism or animal includesthose organisms or animals that have developed from a fertilized egg,into a chromosome of which a foreign gene has been inserted. Suchtransgenic organisms and animals carry the foreign gene insert in everycell. Transgenic organisms and animals are created by using knowntechniques (see, e.g., Sambrook, supra and BIOCHEMISTRY WITH CLINICALCORRELATIONS (T. Devlin ed., 3d ed. 1992), which is incorporated hereinby reference in its entirety for all purposes). Transgenic organisms andanimals can be used to study different aspects of the foreign gene,including the analysis of DNA regulatory elements, expression ofproteins during differentiation, tissue specificity, and the potentialrole of oncogene products on growth, differentiation, and the inductionof tumorigenesis. A “transgene” is a gene, in original or modified form,that has been introduced into an organism or animal that does notnaturally have such gene. A “mosaically expressing transgene” is atransgene that is expressed randomly in a subset of the cells of thetransgenic organism or animal. An “exogenous gene” is a gene from anorganism or animal that does not belong to the species into which thegene has been introduced. A “transient transgenic animal” is transgenicanimal which carries an introduced gene that is not inserted into achromosome.

The term “founder fish” as used herein refers to the fish from which aline of fish is generated. Usually, a founder fish is an individual fishwhich carries a unique mutation and which is used to generate progenythat also carry the mutation.

A “physiological activity” in reference to an organism is defined hereinas any normal processes, functions, or activities of a living organism.

A “prophylactic activity” is an activity of, for example, an agent,gene, nucleic acid segment, pharmaceutical, substance, compound, orcomposition which, when administered to a subject who does not exhibitsigns or symptoms of a disease or exhibits only early signs or symptomsof a disease, diminishes, decreases, or prevents the risk in the subjectof developing pathology.

A “therapeutic activity” is defined herein as any activity of e.g., anagent, gene, nucleic acid segment, pharmaceutical, therapeutic,substance, compound, or composition, which diminishes or eliminatespathological signs or symptoms when administered to a subject exhibitingthe pathology. The term “therapeutically useful” in reference to anagent means that the agent is useful in diminishing, decreasing,treating, or eliminating pathological signs or symptoms of a pathologyor disease.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

I. General

The present invention is directed to methods of screening an agent foran activity. In Section II of the application, methods of screening anagent for an ability or capacity to enhance, inhibit, or blockangiogenesis activity are discussed. In Section III, methods ofscreening an agent for an ability to enhance, inhibit, or cause celldeath activity are described. In Part IV, methods of screening an agentfor a toxic activity are presented.

A. Animal Models

The methods of the present invention, which are directed to screeningagents for activities (e.g., angiogenesis activity, cell death activity,and toxic activity), are generally applicable for use in a variousanimals, including vertebrate animals, such as fish. Various species offish are suitable, including teleosts. Suitable teleosts include, forexample, zebrafish (Danio rerio), Medaka, Giant rerio, and puffer fish.Typically, animal models of the present invention are fish that aretransparent or translucent (i.e., optically clear) in at least one ofthe following stages: the embryonic, larval, or adult stage.

Certain teleosts, including the zebrafish, Medaka, Giant rerio, andpuffer fish, offer important advantages over other animal model systemsfor use in screening methods of the present invention. First, theseteleosts are vertebrates whose genetic makeup is more closely related tothat of man than are other models, such as the Drosophila and nematode.All essential components of human form and organ development aremimicked in these teleosts and the morphological and molecular bases oftissue and organ development are either identical or similar to othervertebrates, including man. Chen and Fishman, Development 123:293-302(1996); Granato and Nusselien-Volhard, Cur. Op. Gen. Dev. 6:461-468(Wylie ed. 1996). As a result, these teleosts serve as an excellentmodel for the study of vertebrate development and human disease states.

Second, these teleosts provide advantageous animal models because theirembryos are very transparent. Given the transparency of the embryo,angiogenesis activity, cell death activity (e.g., apoptosis andnecrosis), and toxic activity produced by administered agents can bedetected and diagnosed much more rapidly than in non-transparentanimals. These activities can also be detected in the more mature larvaland adult forms of the zebrafish, though somewhat less readily as suchforms become progressively less optically clear. These activities canalso be detected in vivo in all three forms or in cells thereof invitro. By contrast, the mouse, which is commonly used as an animal modelsystem, is an opaque animal and does not allow a similar rapid or invivo assessment of phenotypic or developmental changes, including thoseassociated with cell death, angiogenesis, or toxicity, in whole animalor whole organs or tissues. Significantly, precursor tissues andcomponents of the brain, eyes, heart, and musculature of these teleostsare detected and visualized much more easily and quickly in thetransparent teleosts than in other systems, including other vertebratesystems (such as the mouse) by a variety of detection techniques,including, e.g., light microscopy, fluorescence microscopy, colorimetry,chemiluminescence, digital imaging, microplate reader techniques, insitu hybridization of RNA, etc.

Another important advantage of teleosts over other animal models is thatteleosts develop much more rapidly than do other animal models. Ingeneral, the body plan, organs, tissues, and other systems of teleostsdevelop much more rapidly than do such components in other vertebratemodel systems (e.g., the mouse). The entire vertebrate body plan of thezebrafish, for example, is typically established within 24 hours. Afunctioning cardiovascular system is evident in the zebrafish within thefirst 24 hours of development. Stainier and Fishman, Trends Cardiovasc.Med. 4:207-212 (1994). The remaining organs of the zebrafish, includingthe gut, liver, kidney, and vasculature, are established within 48hours. The hatched zebrafish embryo nearly completes morphogenesiswithin 120 hours, thereby making it highly accessible to manipulationand observation and amenable to high-throughput automated observationand detection procedures.

The cell death activity, angiogenesis activity, and toxic activity of anagent and responses indicating these activities can be monitored inwhole teleosts and/or in vivo or in cells thereof in vitro over time—aprocedure not possible or readily practiced with other animal embryoswhich develop in utero, such as the mouse. Moreover, the effects of anagent on the whole teleost embryo or on more than one system (e.g.,cardiovascular system, enteric system, and musculature system), organ,or tissue can be detected simultaneously using transparent teleosts. Thepersistence of such effects can be monitored by using simplevisualization methods and over selected time intervals. By comparison,it is extremely difficult to detect and assess developmental andphenotypic changes in organs, tissues, and systems (such as inhibitionor enhancement of angiogenesis, cell death or toxic activity due to anagent) over time in animals which develop in utero. Mouse embryos, forexample, must be removed from the mother—a labor intensiveprocedure—before an assay can be performed.

Teleosts also offer the advantage that agents to be evaluated for toxiceffects can be administered directly to the developing teleost. Directintroduction of candidate compounds is hindered in animals which developin utero, such as the mouse embryo. Further, the teleost embryo is anintact, self-sustaining organism. It is different from a mouse embryo,for example, which because it is physically removed from its mother'swomb, it is not self-sustaining or intact; a mouse embryo would functionmore as an “organ” culture or the like.

Another significant advantage is cost. Mouse assays are expensive,primarily due to the cost of breeding and maintenance and the need tomanually perform injections and subsequent analysis. The average cost ofa commercial mouse tumor assay is approximately $2,900 ($1,600 pergovernment). In contrast, teleosts, such as zebrafish, are comparativelyinexpensive to generate and maintain. For example, the estimate cost ofa zebrafish assays is less than $100. A single mating of a zebrafishproduces 100-200 eggs. Inbred strains are available and thousands ofzebrafish can be raised inexpensively in a small room of aquaria.Moreover, teleost eggs, including those of the zebrafish, are externallyfertilized. Teleost embryos (such as zebrafish) can survive by diffusionof oxygen from the water and nutrients from the yolk and thus even theabsence of the entire circulatory system is well tolerated during earlydevelopment. Weinstein et al., Nature Med. 1:1143-1147 (1995).

Additionally, single whole teleost embryos can be maintained in vivo influid volumes as small as 100 microliters for the first six days ofdevelopment. Intact live embryos can be kept in culture in individualmicrotiter wells or multi-well plates. Test compounds can be addeddirectly to the solution in which the fish is immersed. Compoundspermeate the intact embryo directly, making this multi-well formatparticularly attractive for high through-put and automated compoundscreening. Both the therapeutic activities and side effects (e.g.,toxicity) of a drug can be assayed in the fish simultaneously in vivo.

The teleosts used with the screening methods of the invention aretypically early-stage teleost embryos; however, transparent larval oradult teleosts can also be used. Wildtype strains of teleosts areusually employed. Wildtype strains are typically maintained for aboutone year, after which time fertility decreases. Mutant strains ofteleosts (such as zebrafish) can be used to assess, e.g., theinteraction between therapeutic agents and specific geneticdeficiencies. The teleost can contain a mutation in a selected gene. Themutation can be a heritable mutation, including, e.g., a heritablemutation associated with a developmental defect. The teleost can also betransgenic.

B. Agents to be Screened

A variety of agents from various sources can be screened for enhancingor inhibiting angiogenesis activity, cell death activity, and/or toxicactivity by using the methods of the present invention. Agents to bescreened can be naturally occurring or synthetic molecules. Agents to bescreened can also obtained from natural sources, such as, e.g., marinemicroorganisms, algae, plants, fungi, etc. Alternatively, agent to bescreened can be from combinatorial libraries of agents, includingpeptides or small molecules, or from existing repertories of chemicalcompounds synthesized in industry, e.g., by the chemical,pharmaceutical, environmental, agricultural, marine, cosmeceutical,drug, and biotechnological industries. Agents can include, e.g.,pharmaceuticals, therapeutics, environmental, agricultural, orindustrial agents, pollutants, cosmeceuticals, drugs, organic compounds,lipids, glucocorticoids, antibiotics, peptides, proteins, sugars,carbohydrates, chimeric molecules, etc.

Combinatorial libraries can be produced for many types of compounds thatcan be synthesized in a step-by-step fashion. Such compounds includepolypeptides, proteins, nucleic acids, beta-turn mimetics,polysaccharides, phospholipids, hormones, prostaglandins, steroids,aromatic compounds, heterocyclic compounds, benzodiazepines, oligomericN-substituted glycines and oligocarbamates. Large combinatoriallibraries of compounds can be constructed by the encoded syntheticlibraries (ESL) method described in Affymax, WO 95/12608, Affymax WO93/06121, Columbia University, WO 94/08051, Pharmacopeia, WO 95/35503and Scripps, WO 95/30642 (each of which is incorporated herein byreference in its entirety for all purposes). Peptide libraries can alsobe generated by phage display methods. See, e.g., Devlin, WO 91/18980.Compounds to be screened can also be obtained from governmental orprivate sources, including, e.g., the National Cancer Institute's (NCI)Natural Product Repository, Bethesda, Md., the NCI Open SyntheticCompound Collection, Bethesda, Md., NCI's Developmental TherapeuticsProgram, or the like.

C. Administration of Agents

Agents to be screened for an effect on angiogenesis activity, cell deathactivity, and/or toxic activity can be administered to the teleost byadding the agent directly to the media containing the live teleost.Alternatively, the agent can first be dissolved in the media and thelive teleost submerged in the media subsequently. Such approaches havebeen used to introduce anesthetics and other chemicals to fish embryos.See, e.g., M. Westerfield, THE ZEBRAFISH BOOK: A GUIDE FOR THELABORATORY USE OF ZEBRAFISH (3d. ed. 1995), which is incorporated hereinin its entirety for all purposes. Agents can also be administered to theteleost by using microinjection techniques in which the agent isinjected directly into the live teleost. For example, agents can beinjected into either the yolk or body of a teleost embryo or both.

Agents can also be administered to teleosts by electroporation,lipofection, or ingestion or by using biolistic cell loading technologyin which particles coated with the biological molecule are“biolistically” shot into the cell or tissue of interest using ahigh-pressure gun. Such techniques are well known to those of ordinaryskill in the art. See, e.g., Sambrook et al., supra; Chow et al., Amer.J Pathol. 2(6):1667-1679 (1998).

Agents can be administered alone, in conjunction with a variety ofsolvents (e.g., dimethylsulfoxide or the like) or carriers (including,e.g., peptide, lipid or solvent carriers), or in conjunction with othercompounds.

Agents can be administered to the teleost before, at the same time as,or after administration of a dye used for detection of the response inthe animal indicating a specific activity (e.g., cell death activity,angiogenesis activity, toxic activity).

D. Administration of Dyes

A dye used in methods of screening agents for an activity (e.g., celldeath activity, angiogenesis activity, toxic activity) can beadministered to the teleost by adding the agent directly to the mediacontaining the live teleost. Alternatively, the dye can first bedissolved in the media and the live teleost submerged in the mediasubsequently. See, e.g., Westerfield, supra. Dyes can also beadministered to the teleost by using microinjection techniques in whichthe dye is injected directly into the live teleost. Dyes can be injectedinto either the yolk or body of a teleost embryo or both.

Dyes can be administered alone, in conjunction with a variety ofsolvents (e.g., dimethylsulfoxide or the like), or in conjunction withother dyes. Dyes can be administered to the teleost before, at the sametime as, or after administration of a dye used for detection of theresponse in the teleost indicating a specific activity (e.g., cell deathactivity, angiogenesis activity, toxic activity). When fluorescent dyesare used (e.g., unsymmetrical cyanine dye, such as a quinolium dye) fordetection of an activity (e.g., cell death activity), the dye ispreferably administered prior to administration of the agent.

E. Detecting Agent Activity and Responses in Teleosts

A variety of techniques can be used together or separately to generate asignal and to detect and assess the effect of an agent on cell deathactivity or angiogenesis activity or toxic activity of an agent. Signalscan be generated by, for example, in situ hybridization, antibodystaining of specific proteins (e.g., antibody markers that labelsignaling proteins involved in angiogenic vessel formation in teleost,including VEGF and Ang1 and 2; terminal deoxyuridine nucleotide endlabeling to detect dead or dying cells, etc.). Responses indicatingtoxic or angiogenic activity or an effect of cell death activity can bedetected by, e.g., visual inspection, colorimetry, fluorescencemicroscopy, light microscopy, chemiluminescence, digital imageanalyzing, standard microplate reader techniques, fluorometry, includingtime-resolved fluorometry, visual inspection, CCD cameras, videocameras, photographic film, or the use of current instrumentation suchas laser scanning devices, fluorometers, photodiodes, quantum counters,plate readers, epifluorescence microscopes, scanning microscopes,confocal microscopes, flow cytometers, capillary electrophoresisdetectors, or by means for amplifying the signal such as aphotomultiplier tube, etc. Responses can be discriminated and/oranalyzed by using pattern recognition software. Agents are identifiedand selected using the screening methods according to the activities andresponses they produce.

Changes in the distribution of a protein both spatially and temporally,including a complete absence of a protein, can be detected and proteinexpression profiles can be generated. Changes in a level of an enzyme orenzymatic activity within the intact teleost can also be detected byvarious means, including, e.g., alkaline phosphatase staining and use ofstreptavidin (avidin) conjugated reporter enzyme to detect naturallybiotinylated carboxylase enzymes in the liver, gut, and digestive tubeof animals.

F. Automated Methods

In addition to manual screening methods, the present invention alsoprovides methods for rapid screening of agents for activities, such asangiogenesis activity, cell death activity, and toxic activity, usingautomated procedures. Such automated methods can be readily performed byusing commercially available automated instrumentation and software andknown automated observation and detection procedures. Multi-well formatsare particularly attractive for high through-put and automated compoundscreening. Screening methods can be performed, for example, using astandard microplate well format, with a whole zebrafish embryo in eachwell of the microplate. This format permits screening assays to beautomated using standard microplate procedures and microplate readers todetect enhancement or inhibition of angiogenesis activity in thezebrafish embryos in the wells. A microplate reader includes any devicethat is able to read a signal from a microplate (e.g., 96-well plate),including fluorometry (standard or time-resolved), luminometry, orphotometry in either endpoint or kinetic assays. Using such techniques,the effect of a specific agent on a large number of teleosts (e.g.,teleost embryos) in vivo or in vitro can be ascertained rapidly. Inaddition, with such an arrangement, a wide variety of agents can berapidly and efficiently screened for their respective effects on thecells of teleosts contained in the wells.

Sample handling and detection procedures can be automated usingcommercially available instrumentation and software systems for rapidreproducible application of dyes and agents, fluid changing, andautomated screening of target compounds. To increase the throughput of acompound administration, currently available robotic systems (e.g., theBioRobot 9600 from Qiagen, the Zymate from Zymark or the Biomek fromBeckman Instruments)—most of which use the multi-well culture plateformat—can be used. The processing procedure involves a large number offluid changes that must be performed at defined timepoints.Non-automated throughput is typically 5 microtiter plates perinvestigator (400 teleost embryos and 20 compounds) per week based onusing a 96-well plate with 1 embryo per well and screening 2concentrations with 10 embryos per concentration. Using currentlyavailable fluid handling hardware (e.g., Bodhan Automation, Inc.,Zymark) and our standard sample handling procedures, 50-100 plates perday (4800-9600 teleost embryos and 200-400 compounds) can be processed.Incorporation of commercially available fluid handling instrumentationsignificantly reduces the time frame of manual screening procedures andpermits efficient analysis of many agents, including libraries ofagents.

II. Methods of Screening an Agent for an Effect on Angiogenesis Activity

A. Angiogenesis

The formation and establishment of a vascular supply is an essentialrequirement for the cellular inflow of nutrients, outflow of wasteproducts, and gas exchange in most tissues and organs. Two processes forsuch blood vessel development and differentiation have been identified.One process of vascularization, termed “vasculogenesis,” occurs in theembryo and consists of the in situ differentiation of mesenchymal cellsinto hemoangioblasts. Hemoangioblasts are the precursors of bothendothelial cells and blood cells. The second process, termed“angiogenesis,” involves the formation of new blood and lymph vesselsfrom preexisting endothelium. In this process, tissues and organs arevascularized by sprouting in which smaller vessels extend from largervessels and penetrate a specific tissue. Fouquet et al., supra.Angiogenesis also involves the migration and proliferation ofendothelial cells, their differentiation into a tube-like structure, andthe production of a basement membrane matrix around the vessel. Herbertet al., L. Cell. Biol. 106:1365-1373 (1988).

Methods for screening agents for inhibition or enhancement ofangiogenesis activity are useful in identifying agents that would beeffective in therapeutic or prophylactic treatment of a variety ofdiseases involving angiogenic processes.

B. Blood Vessel Formation

New blood vessels form during normal tissue growth and repair in aseries of sequential steps: an endothelial cell which forms the wall ofan existing small blood vessel (capillary) becomes activated, secretesenzymes that degrade the extracellular matrix (the surrounding) tissue,invades the matrix, and begins dividing. Eventually, strings of newendothelial cells organize into hollow tubes, creating new networks ofblood vessels that make tissue and repair possible. Ordinarily,endothelial cells lie dormant. However, when necessary, short bursts ofblood vessel growth occur in localized parts of tissues. New capillarygrowth is tightly controlled by a finely tuned balance between factorsthat activate or inhibit endothelial cell growth. About 15 proteins areknown to activate endothelial cell growth and movement, includingangiopoietins, epidermal growth factor, estrogen, fibroblast growthfactors, prostaglandin, tumor necrosis factor, vascular endothelialgrowth factor (VEGF), and granulocyte stimulating factor (Zetter, Ann.Rev. Med. 49:407-424 (1998)). VEGF binds to tyrosine kinase receptorsflt-1 and flk-1/KDR on endothelial cells (Hanahan, Science277(5322):48-50 (1997)). Downstream effects of VEGF include theactivation of matrix proteases and glucaronidases, loosening ofendothelial cell junctions and proliferation and migration ofendothelial cells. Downstream effects of basic fibroblast growth factor(bFGF) include the mitogenic stimulation of endothelial cells (Relou etal., Tissue Cell 5:525-530 (1998)). Some of the known inhibitors ofangiogenesis include angiostatin, endostatin, interferons, interleukin1, interleukin 12, retinoic acid, and tissue inhibitors ofmetalloproteinase 1 and 2 (Zetter, supra).

C. Angiogenesis Inhibition

Because angiogenesis is essential for solid tumor growth, inhibition ofangiogenesis is one strategy for preventing tumor growth. By blockingthe development of new blood vessels, a tumor's supply of oxygen andnutrients can be cut off and, therefore, the tumors' continued growthand metastasis can be arrested. Several strategies can be to designanti-angiogenesis agents including: 1) blocking the factors thatstimulate the formation of blood vessels; 2) using natural inhibitors ofangiogenesis; 3) blocking molecules that allow newly forming bloodvessels to invade surrounding tissue; and 4) incapacitating newlydividing endothelial cells. In general, tumors with higher densities ofblood vessels are more likely to metastasize and are correlated withpoor clinical outcomes. Also, cell shedding from the primary tumorbegins only after the tumor has a full network of blood vessels. Inaddition, both angiogenesis and metastasis require matrixmetalloproteinases, enzymes that break down the surrounding tissue andthe extracellular matrix during blood vessel and tumor invasion. Severaldifferences between standard chemotherapy and anti-angiogenesis therapyresult from the fact that angiogenic inhibitors target dividingendothelial cells rather than tumor cells. Anti-angiogenic drugs are notlikely to cause bone marrow suppression, gastrointestinal symptoms, orhair loss, characteristics of standard chemotherapy treatments. Also,because anti-angiogenic drugs may not necessarily kill tumors, butrather hold them in check indefinitely, the endpoint of early clinicaltrials may be different than for standard therapies. Rather than lookingonly for tumor response, it may be appropriate to evaluate increases insurvival and or time to disease progression.

Drug resistance is a major problem with chemotherapy agents because mostcancer cells are genetically unstable and therefore prone to mutations.Because angiogenic drugs target normal endothelial cells, which are notgenetically unstable, drug resistance may not develop. So far,resistance has not been a major problem in long term animal studies orin clinical trials of potential therapeutic drug candidates.Anti-angiogenic therapy may prove useful in combination with therapydirectly aimed at tumor cells. Because each therapy is aimed atdifferent cellular targets, such combination therapy should moreeffective. There is growing recognition that cancer may become a chronicdisease. If treatments are long term, the toxicity profile of drugs,which can be examined readily in the transparent teleost (e.g.,zebrafish) embryo, will become an increasingly important parameter fordrug screening and evaluation.

D. Angiogenesis Stimulation

Although ischemic tissue in the heart or limbs secretes VEGF and bFGF,which stimulate local growth of collateral blood vessel, naturalformation of collateral vessels feeding into ischemic tissue is rarelysufficient for full restoration of blood flow in cardiovascular diseasepatients. Growth of new blood vessels, induced by exogenous angiogenicagents, may restore blood flow to ischemic tissue in patients withvarious cardiovascular diseases. Stimulatory angiogenic therapy may alsoprovide a minimally invasive approach to improved circulatory functionin coronary artery disease (CAD), congestive heart failure, peripheralarterial disease (PAD) and peripheral venous disease (PVD). Stimulatoryangiogenic therapies may also facilitate transplant acceptance orsurvival. Disadvantages of angiogenic stimulators include exacerbationof growth of occult tumors and progression of diabetic retinopathy. Anideal angiogenic agent for inducing growth of collateral arteries aroundan atherosclerotic plaque should function only in the locality of or bedelivered locally to ischemic tissue.

Angiogenesis gene therapy is an experimental technique being used totrick the heart into performing its own bypass operation by growing newblood vessels. The gene coding for a protein, such as VEGF, whichencourages new blood vessels to sprout from existing ones is injectedinto the heart of the patient and the body performs its own coronarybypass. These new vessels are less inclined to silt up again. Inpreliminary experiments with rabbits, the arteries in rabbit legs havebeen tied off and the VEGF gene has been applied directly onto thesmooth muscle cells lining the artery using a catheter and smallballoon. Within three to 10 days, new blood vessels were observed tosprout and find their way around the blockage. Rivard et al.,Circulation 99(1):111-120 (1999). In preliminary experiments withhumans, the gene has been injected directly into the left ventricle, thepumping chamber of the heart. Results to date from these studies arepromising. There have been no side effects and the worst result to datehas been no result. The sprouting of new vessels, if it occurs, seems tostop after four to six weeks. Losordo et al., Circulation98(25):2800-2804 (1998).

E. Angiogenesis in Zebrafish

In the zebrafish, as in other vertebrates, blood vessels form fromprecursors cells (angioblasts) distributed widely throughout themesoderm of the embryo. Some angioblasts migrate long distances, whileothers remain locally to form vessels (Fouquet et al., supra). The majorvessels, including the aorta, vena cava, and vessels directed to someorgans, are believed to form by local assembly of angioblasts into tubes(vasculargenesis). See FIG. 1. In addition to vasculargenesis, smallervessels extend from larger vessels to penetrate a specific tissue(angiogenesis) (Fouquet et al., supra). Experiments suggest that bothprocesses of vessel formation—vasculargenesis and angiogenesis—aredriven by local signals. By day three of development, the zebrafish hasdeveloped an intact, functioning vasculature, including both majorvessels and sprouts, which has a consistent pattern of vessel location.See FIGS. 2A and 3A. Because the zebrafish embryo can survive anddevelop for at least 4-5 days without a circulatory system, with thetransparent zebrafish it is possible to study the effects of a varietyof agents on all aspects of vascular formation in an intact, liveanimal.

F. Advantages of Using Zebrafish in Screening Assays for Angiogenesis

Currently, a variety of assays are used to study the process ofangiogenesis in various animal models. These assays include preparing atransparent window in the skin of a rabbit or mouse, injecting tumorcells or carrier matrix into an avascular region, such as the cornea,and inducing ischemia by constricting existing blood vessels (Jain etal., Nat. Med. 11:1203-1208 (1997)). While these and other approachesgenerate a great deal of information about the process of angiogenesis,the tissue manipulation required for each assay make them unsuitable foruse as screening tools. (Comparative assays are further described indetail below.) Teleosts and zebrafish in particular offer significantadvantages for in vivo screening assays for angiogenesis. As notedabove, zebrafish are comparatively inexpensive to generate and maintainand the embryos can be placed in individual microtiter wells, makingautomated analysis with standard liquid handling equipment possible.

In addition, with teleosts, such as zebrafish, the side effects of anagent can be monitored and assessed simultaneously along with theprincipal effect of the agent. This provides a significant advantage inmethods for screening compounds for angiogenesis activity. Notably, onedifficulty associated with identifying compounds that can be used asanti-angiogenic agents, such as anti-cancer therapeutics, is that manyof the compounds used to inhibit the proliferation of cancer cells alsohave deleterious effects on proliferating non-cancer cells. This isespecially problematic when dealing with cancers that affect children,because many of their organs and tissues are still growing anddeveloping. Using transparent teleost embryos, the effect of an agent onangiogenesis activity as well as any toxic or side effects can beassayed simultaneously. Side effects or toxic effects of agents onzebrafish cells and/or embryogenesis can be monitored at time intervalsafter administration of the agent. Typically, measurements are performedat the same time as measurements to assess activity of administeredagents.

G. Angiogenesis Screening Methods

The present invention provides methods of screening an agent for anability or capacity of an agent to enhance, inhibit, or blockangiogenesis activity in a teleost in response to the administration ofa dose of an agent to the teleost. Angiogenesis activity is assessedrelative to contemporaneous and/or historical control teleosts (ortissues, organs, or cells thereof) to which the agent has not beenadministered. Angiogenesis activity is reflected in changes in thevasculature of the teleost. Blood vessel formation and development canbe monitored over time in the teleost to which an agent has beenadministered as well as in control teleosts. A response showing anincrease in normal blood vessel formation suggests that the compoundenhances or increases angiogenesis. A response showing a decrease orreduction in normal blood vessel formation or the death or loss ofpreviously established, existing blood vessels suggests that thecompound decreases, prevents, or inhibits angiogenesis activity (i.e.,enhances or stimulates anti-angiogenesis activity) or disrupts existingvessels. Responses indicating an angiogenic activity can be detected ina whole teleost or in one or more organs or tissues of a teleost, eithersimultaneously or separately. Responses can be detected over time and atpredetermined time intervals. These responses can also be detected invitro in cells of a teleost.

The methods of the present invention are useful in identifying agentsthat would be effective in therapeutic or prophylactic treatment of avariety of diseases involving angiogenic processes, including cancer,coronary artery disease, congestive heart failure, peripheral arterialdisease, peripheral venous disease, neurological diseases,cardiopulmonary diseases, ischemia, developmental diseases, autoimmunediseases, and diseases of bone and cartilage. In general, these methodsare useful in screening compounds for therapeutic activity againstdiseases that would benefit from an increase in angiogenesis activity(e.g., increase in blood vessel formation) or decrease in angiogenesisactivity (i.e., anti-angiogenesis activity, a reduction in blood vesselformation).

In one aspect, the methods comprise administering the compound to bescreened to a teleost embryo by submerging the embryo in culture mediain which the compound has been dissolved prior to the onset ofvasculargenesis or angiogenesis. After a suitable period (e.g., 24 or 48hours), the embryos are fixed and stained for an endogenous blood vesselmarker, such as, e.g., alkaline phosphatase (AP). A reduction orincrease in the formation of blood vessels and any perturbation in thenormal pattern of blood vessels can be determined visually by lightmicroscopy after, e.g., alkaline phosphatase staining, antibody stainingof a protein, in situ hybridization. Organ or tissue function can alsobe determined by measuring enzymatic activity.

Compounds comprising small molecules typically penetrate the teleostembryos by simple diffusion. For compounds that do not penetrate theperiderm (the outer ectoderm), dimethyl sulfoxide (DMSO) or othersolvents or osmotic shock can be used to transiently premeabilize theperiderm. Compounds can also be administered by other well-known methodsof administration, including ingestion or direct injection into eitherthe embryo yolk or the heart of the teleost embryo. Once inside theembryo, compounds diffuse freely within the embryo.

For example, to screen for an effect of the compounds on angiogenesisactivity, the subintestinal and intersomitic vessels are typicallyexamined. To screen for an effect of the compounds on vasculargenesisactivity, the dorsal aorta and ventral vessels are examined. All ofthese vessels are quite prominent in the unaffected teleost embryo andthus serve as ideal indicators of changes in the vascular pattern. Inparticular, these vessels are examined for: 1) the presence or absenceof vessels, which is indicative of inhibition of angiogenesis; 2)excessive branching, which is indicative of enhancement of angiogenesis;and 3) changes in architecture of the blood vessel formation, which isindicative of changes in local signaling events. In our methods, thezebrafish embryo is used because it can survive and develop for about4-5 days without a circulatory system and thus the effects of agents onall aspects of vascular formation in the intact embryo can be readilyevaluated.

Changes in vascular pattern can be studied by performing RNA in situhybridization analysis, to examine the angioblasts and vascular growthfactors, and microangiography, to examine the circulation and heartfunction—all of which have roles in blood vessel formation. As anexample, a compound to be screened is administered to a 24-hour teleostembryo by dissolving the compound in the culture medium containing theembryo in culture (prior to the onset of vasculargenesis orangiogenesis). After an additional 24 hours (at 48 hours ofdevelopment), the embryo is visually inspected for morphologicaldefects. 50% of the embryos are fixed for in situ hybridization usingthe flk-1 probe to identify angioblasts. The remaining embryos are fixedat 72 hours of development and stained with AP. Compounds that affectthe expression of endogenous AP, thereby making it difficult to assayvascular pattern by using AP staining, can be assayed by usingmicroangiography. The embryos are then examined for any perturbation inthe normal pattern of blood vessels.

Angiogenesis activity can also be detected by standard techniquesindicated previously, including, e.g., colorimetry, fluorescencemicroscopy (including, e.g., time-resolved fluorometry),chemiluminescence, digital image analyzing, standard microplate readertechniques, pattern recognition software for response discrimination andanalysis, etc. Antibody staining of specific epitopes can also be usedto detect spatial or temporal changes in distribution and expression ofepitopes in teleost tissues, as well as molecular modifications.

H. Screening Agents for Angiogenesis Activity and/or Toxic Activityand/or Cell Death Activity Simultaneously

The methods for screening agents for angiogenesis or anti-angiogenesisactivity can be combined with other methods of the present inventiondescribed below, including methods of screening agents for an effect oncell death activity (Section III) or toxic activity (Section IV).Because the teleosts used with these methods are transparent, it ispossible to assess angiogenesis or anti-angiogenesis activity inconjunction with other activities. Responses indicating variousactivities can also be detected in conjunction with one another—eitherat separate times or simultaneously.

Such combined methods are useful in assessing multiple affects of anagent on a teleost. The agent may cause both a desired response, such asenhancement of angiogenesis, and a toxic (undesired) response. Theability to assess multiple activities and responses in a teleost due tothe administration of an agent is of particular benefit in identifyingpotential therapeutic compounds and assessing their side effects. Forexample, one difficulty associated with identifying compounds that canbe used as anti-cancer therapeutics against targeted cancer cells isthat some compounds may also have deleterious effects on non-cancercells. Anti-angiogenic cancer therapy, for example, typically seeks toinduce apoptosis in cancer cells by cutting off the blood supply of suchcells. This type of treatment regime may be designed to induce apoptosisin the angioblasts as a means of preventing or diminishingvascularization of the tumor. During treatment, a balance must beachieved such that a negligible level of cell death is induced in othertissues or locations in the body (such as the heart). Such undesiredectopic cell death could be considered a toxic activity. A combinationscreen for assessing angiogenesis, cell death, and toxic activities ofan agent would be useful in identifying those agents that protect theheart from agents which induce apoptosis elsewhere. Dose levels of theagent effective to promote one activity without promoting the other canalso be ascertained. Such combined screens would also be useful inidentifying and evaluating agents for pro-angiogenic therapies whichtypically have the therapeutic goal of preventing cell death in adamaged or transplanted tissue.

Multiple activities/responses can be monitored in the whole teleost orin one or more tissues or organs of the teleost. Such activities andresponses can be monitored over time and at predetermined timeintervals. A variety of techniques can be used together or separately toanalyze multiple activities and responses, including, e.g., fluorescencemicroscopy, light microscopy, digital image analyzing, standardmicroplate reader techniques (colorimetry, fluorometry, includingtime-resolved fluorometry, and chemiluminescence), in situhybridization, antibody staining of specific proteins, changes inprotein distribution temporally and spatially within the animal, changesin a level of enzymatic activity in the whole teleost, or tissues,organs or cells of the teleost, etc. Furthermore, the response can bediscriminated and/or analyzed by using pattern recognition software.

In one aspect, the present invention provides a method of screening anagent for an increase or decrease in angiogenesis activity as describedabove which further comprises screening the agent for an increase ordecrease in toxicity by detecting a response in the teleost indicatingan increase or decrease in toxic activity. Such a method is useful,e.g., in identifying contra indications to therapeutic value of acompound.

In another aspect, the invention provides a method of screening an agentfor an increase or decrease in angiogenesis activity as described abovewhich further comprises screening the agent for an ability to enhance orinhibit cell death activity by detecting a response in the teleostindicating an enhancement or inhibition of cell death activity. Such amethod is useful, for example, in identifying contra indications totherapeutic value of a compound. Such combination screens also allow forthe identification of agents which protect the heart from circulatingagents which induce apoptosis elsewhere.

EXAMPLES

1. Screening Compounds for Angiogenesis Activity in Zebrafish

A. Materials and Methods

1) Embryo Collection

Zebrafish embryos were generated by natural pair-wise mating asdescribed in Westerfield, supra, which is incorporated herein byreference in its entirety for all purposes. Four to five zebrafish pairswere set up for each mating; on average, 100-150 embryos per pair weregenerated. Embryos were collected and placed in egg culture mediaprepared by combining 5 grams (g) of Instant Ocean Salt with 3 g ofcalcium sulfate in 25 liters of distilled water at 27° C. forapproximately 20 hours (21 somite stage) before being sorted forviability, using both morphology and developmental stage as criteria.Healthy embryos were then dechorionated by enzymatic digestion using 1mg/ml protease (Sigma Chemistry Co.) for 5 minutes at room temperature.The embryos were then washed 5 times in embryo water. Because the fishembryo receives nourishment from an attached yolk ball, no additionalmaintenance was required.

2) Compounds Screened

Compounds from the following two sources were screened for an ability orcapacity to enhance or inhibit angiogenesis activity: NCI Open SyntheticCompound Collection library, Bethesda, Md. and The Center for CancerResearch, Massachusetts Institute of Technology (MIT), Cambridge, Mass.

The NCI Open Synthetic Compound Collection library consists of more than100,000 unique compound structures; currently, only 12,000 are availablefor screening.

Compounds obtained from MIT consisted of 11 fumagillin derivatives,including TNP 470 (Turk et al., Bioorg. Med. Chem. 8:1163-1169 (1998))and AGM-1470. Fumagillin is a natural product isolated from fungus withpotent anti-angiogenic and toxic effects. AGM-1470 and the otherfumagillin derivatives are angiogenesis inhibitors, which prevent entryof normal, but not transformed, endothelial cells into the G1 phase ofthe cell cycle by binding type 2 methionine aminopeptidase (MetAP2). Thederivatives were supplied at an initial concentration of 20 mM. Sampleswere diluted in dimethyl sulfoxide (DMSO, Sigma Chemical Co.) to a stockconcentration of 10 mM.

Compounds from NCI were randomly selected from the NCI Open SyntheticCompound Collection library. The compounds were supplied by NCI in 96microplate arrays, each at an initial concentration of 10 mM in DMSO. Nospecific information on compound source, activity, chemical structure,or mechanism of action was available.

3) Administration of Compounds

To determine the effect(s) of a compound on vessel formation on a fish,the compound was added directly to the culture medium solutioncontaining the fish embryos (e.g., to individual microwells containingthe fish embryos). Compounds were added to the medium solution at 12 or24 hours of development of the fish embryo, which is prior to the pointat which angiogenic vessels can first be identified using the flk-1 insitu hybridization probe. Fouquet et al., supra. Assays were performedin 6-well, 24-well, or 96-well plates. Such plates facilitatedautomation of the chemical application and subsequent analysis,including dose response, and subsequent analysis.

4) Visual Screening

After administering a compound to the fish embryos, the embryos weremaintained in individual microwells at 28° C. until day 3 ofdevelopment. Twenty-four and forty-eight hours after adding the compoundto the medium in which the fish embryos were cultured, the embryos werevisually inspected for viability, gross morphological defects, heartrate, and circulation (see Table 1). Circulation was assayed byfollowing the movement of blood cells through each embryo.

5) Vessel Staining

On the third day of development, embryos were collected for alkalinephosphatase staining. Specifically, embryos were fixed in 4%paraformaldehyde and stained for endogenous alkaline phosphataseactivity. Embryos were fixed for 2 hours at room temperature. Theembryos were then washed two times in phosphate buffered saline (PBS)and dehydrated by immersion in 25%, 50%, 75% and 100% methanol inphosphate buffered saline with 0.1% Tween (PBT) to permeabilize theembryos. The embryos were then rehydrated and washed in 100% PBT. Forstaining, embryos were equilibrated in NTMT buffer (0.1M Tris-HCl pH9.5; 50 mM MgCl; 0.1M NaCl; 0.1% Tween 20) at room temperature. Afterthe embryos equilibrated, embryos were stained by adding 4.5 μl of 75mg/ml nitro blue tetrazolium (NBT) and 3.5 μL of 50 mg/ml X-phosphateper ml. After staining for 10 minutes, all the blood vessels in the fishembryo were labeled (see FIGS. 2A-2C, 3A-3B, 4, 7). The stainingreaction was stopped by addition of PBST. Embryos were then examined ona stereo-dissecting microscope. One advantage of using the zebrafish forthis type of assay is that the subintestinal vessels, which are locatedover the yolk, are both sensitive to factors which effect vesselformation and easily assayed by this method (see, e.g., FIG. 7). Thesubintestinal vessels are normally present on the dorsolateral surfaceof the yolk of zebrafish embryos by 48 hours of development. They form adistinct basket shape that extends 50-100 μm from the ventral edge ofthe somite over the yolk. By assaying the subintestinal vessels at 72hours of development (24 hours after the subintestinal vessels normallyappear), normal variation in the timing of the vessel formation wasavoided. The staining procedure is easily automated using commerciallyavailable instrumentation.

6) Bleaching Teleosts

If desired, teleosts (e.g., zebrafish embryos) can be bleached before orafter alkaline phosphatase staining. Bleaching removes the melaninpigment from the teleost and permits the screening of teleost withoutthe adverse effects of 1-phenyl-2-thiourea (PTU) treatment. Post-stainbleaching also removes the extracellular staining associated withbackground staining. Bleaching effectively enhances visualization andanalysis of the response of the treated teleost to a compound throughthe removal pigmentation of some cells. Bleaching enhances visualdetection of responses indicating toxic, angiogenic, and cell deathactivities.

To bleach zebrafish, the fish were immersed for 10 minutes at roomtemperature in 5% formamide, 1× sodium chloride/sodium citrate and 10%hydrogen peroxide.

7) In Situ Hybridization

In addition to performing visual screens, specific molecular changes inteleost tissues can be detected by in situ hybridization of RNA orantibody staining of specific proteins. In situ hybridization of RNA isa routine molecular approach in zebrafish (Westerfield, supra). Adigoxigenin-labeling kit from Boehringer Mannheim can be used to labelthe RNA probes. Whole mount in situ hybridization can be carried out asfollows: Embryos are fixed with 4% paraformaldehyde in PBS, lightlydigested with proteinase K, and hybridized with 1 μg of probe in in situhybridization solution (50% formamide, 5×SSC, 50 μg/ml Heparin, 500μg/ml tRNA, 92 μl of 1M citric acid, pH 6.0, and 0.1% Tween 20) at 65°C. Alkaline phosphatase-conjugated anti-digoxigenin antibody is used todetect signals. Background staining from endogenous alkaline phosphatasedoes not pose a problem, because endogenous alkaline phosphatase doesnot survive the in situ hybridization procedure. After staining withNBT/X-phosphatase (Boehringer Mannheim), embryos are bleached in 100%methanol, refixed in 4% paraformaldehyde, and stored in PBS. Multiple insitu hybridizations can be performed simultaneously on differentteleosts in multi-well dishes.

8) Additional Assays for Angiogenesis

To determine if any changes in vascular pattern are due to inhibition orstimulation of the angioblasts, RNA in situ hybridization analysis onknown angioblast markers, flk-1, tie, tek, and fli (Dumont et al., Dev.Dyn. 203:80-92 (1995); Liao et al., Dev. Suppl. 124:381-389 (1996);Fouquet et al., supra) can be performed using procedures outlined above.Flk-1 (FIG. 8), tie, and tek are receptor tyrosine kinases, which labelangioblasts early in development. Fli is a transcription factor whichlabels them at a later stage. Because flk-1, tie, tek, and fli appearsequentially during angioblast development in vertebrates (Dumont etal., supra), assaying for the presence or absence of these moleculesmakes it possible not only to determine if the angioblasts are affected,but also the stage of development at which they are affected.

Changes in the distribution of a protein both spatially and temporally,including a complete absence of a protein, within the intact teleost canbe detected. For example, changes in the pattern of the vascularendothelial growth factor, VEGF, can be examined using standard antibodystaining procedures (Westerfield, supra) or in situ hybridizationtechniques described above (see also Westerfield, supra). VEGF isbelieved to have two roles in vascular development: 1) achemo-attractant or guidance role; and 2) a maintenance role (Dumont etal., supra). Thus, chemicals which affect VEGF expression are ofparticular interest. The above are examples of well known molecularmarkers; other molecular markers can also be employed.

9) Function Assay

In addition to changes in the vascular architecture, vascular function(circulation and heart rate) may also be affected by compounds. Todetermine whether a compound administered to zebrafish affected vascularfunctioning (e.g., heart rate and circulation), heart rate andcirculation of the zebrafish embryos are studied. In this instance,heart rate was assessed by counting the heart beats/minute. Circulationwas assessed by examining zebrafish embryos under a dissectingmicroscope for the movement of blood cells through the heart and majorvessels. Zebrafish embryos were also examined for blood pooling in theyolk (an indicator of poor blood flow through the heart) and in the bodyof the embryo (an indication of leaky vessels). In those embryos inwhich a compound was observed to affect blood cell development,micro-angiography was performed using the procedures outlined inWeinstein et al., Nature Med. 1: 1143-1147 (1995) to examine theintegrity of the vascular system for vessel leakage and blockage, whichcan cause changes in vessel formation and maintenance. Embryos wereanesthetized with tricaine to stop the heart, a micro-pipet was insertedinto the heart, and fluorescent beads were injected. The tricaine wasthen washed out, and the heart resumed beating. The flow of fluorescentbeads was then observed using an epifluorescence microscope and recordedusing a low light level camera attached to a computer (FIG. 9). Thisapproach allows examination of the integrity of the vascular system andassessment of the effects of the chemicals on the condition of theheart.

B. Results

1) Determination of Parameters for the Delivery of Compounds to TargetTissues and Organs

a) Embryo Developmental Stage

In our initial studies, we employed 12-hour zebrafish embryos (6 somitestage) for the assays and began the assays at the 12^(th) hour ofdevelopment. Although this time point is advantageous because it is justprior to the onset of angioblast formation (Fouquet et al., supra),there are several disadvantages. The most significant of these is thatat 12 hours of development, many structures of the zebrafish embryoincluding the notochord, the somites, and the heart are beginning toform. Because these structures directly affect both vasculargenesis andangiogenesis, it is difficult to determine if the observed effects ofcompounds on vessel formation are primary (direct effects on thevessels) or secondary (indirect effects due to damage to other tissues).

To circumvent this problem, we began the assays at 22 hours ofdevelopment (26 somite stage). At this stage of development, the dorsalaorta and ventral vein are present in the anterior, but not in theposterior regions of the zebrafish embryo. This permitted examination ofboth vasculargenesis and angiogenesis independently in the same embryo.For vasculargenesis, we examined the embryos for the presence of thedorsal aorta and ventral vessel in the most posterior regions of thetail. For angiogenesis, we examined the embryos for the presence ofsprouting vessels, including the subintestinal and the intersomiticvessels. The subintestinal vessels begin to form at 36 hours ofdevelopment; therefore, using the 22-hour time point reduces the timebetween compound administration and angiogenic vessel formation. This isan important consideration for compounds that are unstable under theculture conditions.

b) Embryo Maintenance

Initial experiments were performed in 35 mm wells in 6-well culturedishes using 50 zebrafish embryos per well in 5 ml of embryo water.While this approach worked, it has a number of drawbacks, includingthat: 1) a relatively large amount of compound must be used to dose theembryos; 2) the number of compounds that can be screened simultaneouslyis limited; and 3) because there are multiple embryos in a dish, dyingembryos could contaminate living embryos.

In an attempt to circumvent these drawbacks, we examined two alternativeformats, the 96- and the 24-well plate. Previous observations indicatedthat single zebrafish embryos were capable of surviving and developingnormally in 50-100 μl of embryo water for up to 5 days. Therefore, wecollected, dechorionated and sorted 22 hour embryos into either: 1) 96well plates with one embryo per well in 100 μl; or 2) 24 well plateswith 5 embryos in 500 μl of embryo water. The embryos were allowed todevelop for 72 hours before examination. The embryos were assessed bysize, morphology, and movement. No obvious differences were observedbetween the embryos raised in the microwell plates and control embryosraised in larger containers. The embryos were fixed and stained forendogenous alkaline phosphatase to examine vessel formation. Thestaining pattern in the experimental embryos was identical to thatobserved in the controls. For the manual screen, we preferred the 24well format and used it for all experiments described below.

c) Compound Delivery

In order to optimize the parameters for screening compounds, weperformed a series of experiments using the 11 fumagillin derivativesobtained from MIT and 10 random compounds obtained from the NCI OpenSynthetic Compound Collection library. We knew from our feasibilitystudies that fumagillin inhibited angiogenesis in the zebrafish; we thusdecided to use these compounds as positive controls to verify the assay.We also used the 10 compounds from NCI to verify that the establishedparameters were appropriate for other types of compounds. In general, weexpected that the small molecules would diffuse freely both into theembryo and through the chorion membrane that surrounds the embryo forthe first 2-3 days of development. However, to avoid potential problems,we removed the chorion by enzymatic digestion. This approach is wellestablished and when done properly produces no adverse effects on theembryos.

TABLE 1 Summary of Concentration Effects of Compounds Compound 100 μMEffect 10 μM Effect 1 μM Effect MIT (11) 11/11  Lethal 7/10 Vasculareffects 11/11 Slight Developmental Delay Developmental Delay NCI (10)4/10 Lethal 1/10 Lethal  1/10 Lethal 6/10 Slight 2/10 Chromatic Change10/10 Slight Developmental Developmental Delay Delay 10/10  SlightDevelopmental Delay

d) Compound Concentration

As a primary screen for compound effects, we tested each compound atthree different concentrations to determine which concentration wouldprovide the most information. The concentrations tested were 100 μM, 10μM, and 1 μM. Results are summarized in Table 1. For these experiments,we added 50 μl of 10 mM stock solution to 5 ml of embryo water togenerate a 100 μM solution in 1% DMSO. The subsequent concentrationswere generated by 1:10 and 1:100 dilutions in embryo water; for eachconcentration, DMSO added to 1% of the total solution. The controlsolutions consisted of 1% DMSO in embryo water. Ten embryos per compoundper concentration were tested. Of the 21 compounds tested, 15 (11/11MIT, 4/10 NCI) were lethal at the 100 μM concentration. At the 10 μMconcentration, 7/11 of the fumagillin derivatives had an inhibitoryeffect on angiogenesis. However, while none of the fumagillinderivatives were lethal at 10 μM, they all had a deleterious effect onthe growth of the embryo (FIG. 2A), consistent with previously publishedresults showing that the target of fumagillin derivatives is methionineaminopeptidase (type 2), which plays a role in cell cycle control ineukaryotic cells (Ishikawa et al., J. Exp. Ther. Oncol. 6:390-396(1996); Kria et al., Curr. Eye Res. 10:986-993 (1998).

In contrast to the fumagillin derivatives, at 10 μM the NCI compoundshad no observable effect on vessel formation. However, 1 of the 10 NCIcompounds was lethal at this concentration and 2 of the 10 compoundscaused a chromatic change in the embryos. The chromatic changes were notlimited simply to taking up the color of the compound; one of the NCIcompounds caused the melanocytes to turn purple. As with the fumagillinderivatives, all 9 of the non-lethal NCI compounds caused a slightdevelopmental delay, because the embryos appeared by morphologicalcriteria to be ˜12 hours delayed in development. At 1 μM, 20/21compounds caused developmental delay and 1/21 caused lethality. Theseresults show quite clearly that compounds added to the media werecapable of getting into the zebrafish embryo and inducing an effect.

e) Use of DMSO

One problem with the experimental conditions described above was thatthe control embryos maintained in 1% DMSO in embryo water also showed aslight developmental delay, similar to that observed for all of theconcentrations of the NCI compounds and for the 1 μM concentration thefumagillin derivatives. We repeated the experiments using 10 μM and 1 μMconcentrations of the compounds, respectively, in 0.1% DMSO. The resultswere identical to those in Table 1, except that the developmental delayfor all of the compounds except the fumagillin derivatives at 10 μMconcentration was eliminated. After performing these experiments, wedecided to use 10 μM concentrations with 0.1% DMSO. The resultsindicated that at relatively high concentrations, DMSO has some effecton developing zebrafish. While DMSO does not appear to have any effecton developing zebrafish at lower concentrations, we are aware thatsynergistic effects may occur. Unfortunately, many of the compoundsavailable for screening were only soluble in DSMO or similar solvents.As with any primary screen, positive results will require furtherverification and scrutiny.

2) Assessing the Effects of Compounds on Blood Vessel Formation

After establishing basic assay parameters, we screened compoundsreceived from MIT (11 compounds) and NCI (190 compounds) for effects onblood vessel formation (angiogenesis and vasculargenesis). Embryos werecollected at 20 hours of development and dechorionated. At 22 hours ofdevelopment, the embryos were sorted into 24 well plates with 5 embryosper well in 500 μl of embryo water. The compounds from MIT and NCI wereadded at a concentration of 10 μM. For each compound, 3 sets of embryos(15 total) were screened. For convenience, each set was maintained in aseparate multi-well plate. This permitted testing of 23 compounds/platewith 1 set of controls per plate. At 72 hours of development, embryoswere visually screened for gross morphological defects and cardiacfunction using a dissecting microscope. After the visual screen, embryoswere fixed and stained for endogenous alkaline phosphatase activity inorder to analyze vascular architecture. Experimental results are shownin Table 2 and described below.

TABLE 2 Results of Visual Screen Number Develop- Circulation/ Toxic AtCompounds Compds Vascular mental Axial Cranial Heart Rate 10 μM (Compds)Screened Changes Delay Defects Defects Defects Compd NCI 190 18 16 6 7 613 MIT 11 7 11 3 0 0 0

a) Vascular Changes

To assay vessel formation, embryos were fixed and stained and thevessels were scored as described above. The subintestinal vessels formon the dorsolateral surface of the yolk on both sides of the embryo inthe shape of a basket that extends 50-100 μm from the ventral edge ofthe somite over the yolk. For this screen, anti-angiogenic effects weredefined as either the complete absence of these vessels or the loss ofeither the lateral or dorsalventral vessels of the basket (FIGS. 2B-2C,3B, 4). An angiogenic effect was defined for this screen as anenlargement of the basket beyond 150 μm from the somite. This includesboth increases in size of the entire basket and/or projections from thebasket (FIGS. 6A, 6C, 6D). In addition to the overall basket size, wealso looked for increases in the diameter of the vessels. Normal vesselsare less than 10 μm in diameter. Embryos were also screened for grosschanges in the large vessels, including the dorsal aorta and ventralvein.

Of the 241 compounds tested, 25 (7/11 from MIT and 18/190 from NCI)caused some anti-angiogenic effects (Table 3). Of these, 23/25 wereassociated with various degrees of developmental delay; the more severethe delay, the more dramatic was the reduction in vessel formation(FIGS. 2A-2B). Of the two other compounds that caused a reduction orloss in vessel formation, one was associated with a truncation of theembryonic axis (FIG. 4). Axial defects do not generally cause a loss ofthe subintestinal vessels, suggesting that the vessel effect may bedistinct from the axial effect. Only 1 of the compounds tested showed aspecific effect on vessel formation. With this compound, there was aloss of the subintestinal vessels (FIG. 2B), with no other observableeffects on the embryo.

TABLE 3 Observed Effects of Compounds on Vessel Formation Loss ofComplete Loss Lateral or Increase in Changes Compds Vessel ofSubintestinal Dorsalventral Increase in Vessel in Large (Source) EffectVessels (SIVs) Vessels of SIVs SIVs Diameter Vessels MIT (11) 7/11 4 3 00 5 NCI (190) 18/190 5 13 0 0 3

With 8/25 compounds that caused angiogenic effect, we observed whatappeared to be a failure of the large vessels to organize properly inthe tail. We only observed this effect with compounds that inducedsevere developmental delay. It is likely that the failure of the aortaland ventral vein to organize properly may be a secondary effect.Curiously, none of the compounds screened caused an increase in vesselformation as assayed by increases either in size of the subintestinalvessels or the diameter of the vessels.

To determine if the loss of the subintestinal vessels was due to theloss of angioblast, we performed an in situ analysis on embryos treatedwith the compounds that had previously been shown to cause a reductionof vessel formation. We used a probe against flk-1, a receptor tyrosinekinase that has been shown to play a pivotal role in angiogenic vesselformation (Hanahan, supra). Flk-1 has been shown to be the earliestmarker for angioblast in the zebrafish embryo (Fouquet et al., supra).Normally, flk-1 is highly expressed throughout development in newlyforming vessels and expressed at lower levels in the large vessels aftertheir formation. In the absence of angiogenic vessels, we would notexpect to see flk-1 expression in the somitic and subintestinal region;therefore, we focused on expression of flk-1 in the large vessels.

Embryos were collected at 48 hours of development (24 hours afteraddition of the compounds) because Flk-1 is still highly expressed inthe large vessels at this stage. For 17 of the 18 NCI compounds whichcaused a reduction in the subintestinal vessels, the pattern of flk-1staining appeared normal at 48 hrs of development. Specifically, flk-1staining was present in the dorsal aorta and ventral vein as well as inthe vessels of the head. There was no staining in either theintersomitic space or on the dorsal surface of the yolk, however; thiswas expected, because these vessels did not form. One compound caused aloss of flk-1 staining in the large vessels of the tail, but not in thehead. This compound also caused a truncation of the embryo, a thinningof the tail and heart malformation (FIG. 5). It is probable that theloss of flk-1 staining was part of a more global defect, rather than aspecific anti-angiogenic effect.

b) Developmental Delay

Because a number of the defects included changes in the size and shapeof the embryo, to distinguish between morphological defects anddevelopmental delay, we used three distinct parameters. Normally,zebrafish embryos are staged by the position of the head on the yolkball, the length of the embryo, and the position of the formingmelanocytes. As our 4^(th) criteria, we used the size and shape of thefins to assist in staging the embryos. For this screen, developmentaldelay was defined as at least 12 hours slower than the control embryoson the same multi-well plate. As previously noted, all the fumagillinderivatives caused a substantial developmental delay of at least 24hours (FIGS. 2A-2C). This is probably because the target of thesecompounds is a cell cycle regulating protein (Turk et al., supra).Developmental delay was also observed in 16 of the 190 (8.5%) smallmolecule compounds from NCI. In all cases where developmental delay wasobserved, there was a change in vascular architecture consistent withthe developmental delay (FIGS. 2A-2C). It is likely that compounds thataffect proliferation and growth will also affect angiogenic vesselformation, which requires cell proliferation in order to form newvessels. Eight of these 16 compounds also caused what appeared to be adisorganization of the large vessels in the tail.

c) Axial Defects

There were three typical types of axial defects: 1) bending of the axiseither up or down (NCI 3/6, MIT 3/3); 2) truncation of the axis (NCI2/6); and 3) blebbing of the notochord (FIG. 5)(NCI 2/6). A reduction ofthe subintestinal vessels was observed with only one of the compoundsthat caused an axial defect (FIG. 4).

d) Cranial Defects

Cranial defects were defined as either the disruption of the centralnervous system (CNS) morphology, usually at the midbrain/hindbrainborder, or the presence of cellular debris in the ventricular space ofthe CNS. Seven of the 190 NCI compounds caused cranial defects; however,none affected the subintestinal vessel or the large vessels in the tail.

e) Toxicity

For this specific experiment, we defined toxicity as whole embryolethality by 72 hours of development. Using the previously establishedassay parameters, we predicted that the 10 μM concentration of acompound was unlikely to induce toxicity. Therefore, we were notsurprised that only 5% (6.8% of NCI, 13/190) of the compounds testedwere lethal. Of the 13 lethal compounds, eight killed the embryos within24 hours of application. The remaining 5 compounds caused localized celldeath (4 in the tail and 1 in the head) within 24 hours and whole embryolethality by 72 hours of development. It is possible that at lowerconcentrations these compounds can affect angiogenesis without causingtoxicity; however, this seems unlikely, as the toxic effects were quiteglobal.

3) Assessing Effects on Vascular Function

a) Circulation/Heart Rate Defects

There were a number of compounds causing developmental delay and axialdefects that also caused structural changes in the heart. In general,these effects were consistent with underdevelopment of the heart. Inorder to evaluate function, we restricted our analysis to embryos inwhich the heart appeared relatively normal, as defined by the presenceof an atrium and a ventricle, as well as a heartbeat. 6/190 of the NCIcompounds caused a reduction in the beat rate of the heart. For thisscreen, reduced heart rate was defined as 50% or less than the rate ofcontrols. Because biological and environmental factors cause naturalvariations in the heart rate, the normal heart rate was taken as theaverage heart rate of the 10 embryos in the control wells for eachplate. This was compared to the average heart rate of the embryos in theexperimental well. In 3/6 compounds, pericardial edema and blood poolingover the yolk accompanied the reduced rate. Even though pericardialedema was evident, blood cells moved through the major vessels. Allthree of these compounds caused developmental delay with an associatedreduction in angiogenic vessels; specifically, the subintestinal vesselswere absent. The remaining 3 compounds had no observable effects otherthan reduced heart rate.

None of the non-lethal compounds tested caused an observable reductionin the number of blood cells; thus, it was possible to assay circulationby observing the movement of blood cells through the vessels. As withassessment of heart rate, only embryos with structurally normal heartswere analyzed, because malformed or underdeveloped hearts cannot usuallypump blood. None of the compounds appeared to affect circulation asassessed by lack of blood flow, blood pooling, or leaky vessels.

Circulation was assayed by observing the flow of blood cells through theembryo. Of the 212 compounds tested in this study, none affected theformation of the blood cells; therefore, it was not necessary to performany microangiograms to assay circulation. However, because it isunlikely that this will be the case for all compounds, themicroangiogram technique is typically included as part of the screeningmethods. A microangiogram was performed as part of our initial studieson a zebrafish embryo at day three of development. The microangiogramshows the normal vascular pattern of the zebrafish embryo, including thecranial, intersegmental, and subintestinal vessels. See FIG. 9.

C. Discussion

The above results demonstrate that the teleost (e.g., zebrafish) is aviable model for screening small molecules (e.g., chemical compounds)for effects on vessel formation. Such small molecules not only diffuseinto the embryo, but can also induce specific, observable effects onblood vessel formation.

1) Diffusion of Small Compounds into the Teleost Embryo

One major concern prior to experimentation was whether different typesof small molecules would diffuse into the zebrafish embryos afteraddition to the media. Our initial studies demonstrated that fumagillinand ovicillin were capable of diffusing into the zebrafish embryo.However, these compounds are natural products identified because oftheir ability to diffuse into cells in culture. Of the 201 smallmolecule compounds screened, 81 had some observable effect on zebrafishembryos (70/190 compounds, including 23 which caused color changes (datanot shown), from NCI and 11/11 fumagillin derivatives). These resultssuggest that our initial assumption that small molecules would enter theembryos by diffusion was correct.

2) Advantages of Whole Embryo Screening

One significant advantage of using whole teleost embryos for assays isthe ability to identify effects on multiple targets simultaneously. Inour initial set of experiments, we restricted additional targets toevents that could be visualized without additional staining.Developmental delay was the most useful of these parameters. Unlike withcell culture assays, with the whole embryo assay, we were able toobserve that the 11 MIT compounds caused what appeared to be generalcell proliferation effects, which may or may not be the same asanti-angiogenic effects. This may be due to the binding of type 2methionine aminopeptidase (MetAP2)(Turk et al., supra) or a related cellcycle protein.

We also observed a number of other effects with other compounds. With 6compounds, we observed effects on heart rate in live embryos by visualinspection. Because the heart is quite prominent in the early embryo, itwas possible to observe a slow versus normal heart rate by visualinspection. Two possible mechanisms for this observed effect are: 1) thecompound may affect development of the heart in such a way that theconductivity mechanism required for normal heart beat is absent, or 2)the compound directly antagonizes the conductivity mechanism in a mannersimilar to beta-blockers (Reiter and Reiffel, Am. J. Cardiol.82(4A):9-19 (1998)). We were also able to score cranial defects in 7/201compounds, as well as axial defects in 9/201. In subsequent studies(described below), we used specific antibodies and staining techniquesto analyze the effects of compounds on other organs, including the liverand the kidney, to determine adverse effects of angiogenic compounds.The liver and kidney are highly vascularized; as a result, these organsrepresent potential targets for screening compounds for adverse effectson blood vessel formation.

3) Screening for Anti-Angiogenic Effects

In our first set of screening experiments for anti-angiogenic effects,we examined the effect of fumagillin, a natural anti-angiogenesischemical, on blood vessel formation in the zebrafish embryo. Thecompound was administered by addition to the fish culture media. Thiscompound caused a reduction in angiogenesis, indicated by a reduction ofthe subintestinal and intersomitic vessels (see, e.g., FIG. 7). However,each compound also caused serious complications in the embryo includingpericardial edema, developmental delay, and axial defects. Althoughthese experiments demonstrated the feasibility of the approach for drugscreening, they also underscored the importance of identifying compoundsthat affect angiogenesis selectively. Using the screening parametersdescribed above, we identified two compounds that caused apparentlyspecific anti-angiogenic effects. In addition, we identified 16 othercompounds that caused a reduction of angiogenic vessel formation inaddition to other effects. These results show that the zebrafish embryomodel can be used to screen for compounds that specifically affectangiogenesis and anti-angiogenesis activities.

4) Screening for Angioblast Formation Using Flk-1 Staining

1 of the 18 NCI compounds that caused a reduction in subintestinalvessel formation had an effect on the flk-1 staining pattern. Becauseflk-1 is an early marker for angioblasts, this result suggests that for17/18 compounds, the blocking of angiogenesis is not due to loss ofangioblast, but rather to interference with some other component of theangiogenic pathway. For the one compound that did affect flk-1 staining,it was not clear if the loss of staining was due to a loss ofangioblasts or loss of the flk-1 tyrosine kinase expression. Thisdemonstrates the importance of establishing markers for both angioblastsand the angiogenic pathway (see discussion below).

5) Angiogenic Effects

None of the compounds tested caused an observable increase in vesselformation. Two possible explanations of this observation are: 1) none ofthe compounds tested had angiogenic properties; and 2) the normalzebrafish embryo is refractory to exogenous angiogenic stimulation. Todistinguish between these two possibilities, we performed experiments,described below, in which VEGF was injected into 24 hour embryos. Theseexperiments suggested that increased angiogenesis can be induced in thenormal zebrafish embryo (FIGS. 5A-5C). In order to increase thelikelihood of identifying compounds which stimulate angiogenesis, weexplored the use of mutant zebrafish lines, such as the gridlock mutant(Weinstein et al., supra), which has defects which block angiogenesis.

6) Vasculargenesis

In the zebrafish as in humans, vasculargenesis is the process by whichthe large vessels, including the aorta, vena cava, and vessels to someorgans, form from local precursors cells (angioblasts) distributedthroughout the mesoderm of the embryo (Fouquet et al., supra). Weobserved vascular defects with 8 of the 241 compounds screened. Theeffects observed were limited to a disorganization of the dorsal aortaand ventral vessel in embryos with severe developmental delay. It is notclear that vasculargenesis requires extensive cell proliferation, andthese observed effects on the large vessels may be due to a disruptionof the surrounding tissue, rather than a direct effect on theangioblasts.

7) Rapid and Automated Methods of Screening of Agents for AngiogenesisActivity

Our experiments demonstrated the versatility and value of the teleost asa model for use in detecting, identifying, and analyzing compounds thatinhibit or enhance angiogenesis in vivo and in vitro. With methods ofthe present invention, teleosts (e.g., zebrafish embryos) can be used toscreen large numbers of compounds rapidly for effects on angiogenesis.For example, using the 24 well format and manual techniques for fluidchanges, we screened 241 compounds for a variety of effects at multipletime points. These effects included morphological defects, functionaldefects, and lethality. While these target effects provide a tremendousamount of information, analysis of other targets such as heart rate,circulation, and other organs constitute a secondary level of screeningthat should be performed only on compounds pre-screened for angiogeniceffects. A primary screen for compounds which affect angiogenesis shouldfocus on the stained subintestinal vessels in 72 hour embryos.

The present invention also includes automated methods for rapidscreening of compounds that enhance or inhibit angiogenesis activity inanimal models in vivo and in vitro in cells thereof. Preferred animalmodels include transparent teleosts, such as zebrafish. Any of thecompounds described herein can be screened using automated proceduresdescribed previously, including, e.g., small chemical compounds orlarger biological molecules discussed below.

In our analyses discussed above, we screened 190 compounds from the NCIOpen Synthetic Compound Collection library. Although this libraryconsists of more than 100,000 unique compound structures, currently only12,000 are available for screening. Using the manual screening methodsof the invention, the entire compound library can be screened in twoyears. Incorporation of commercially available fluid handlinginstrumentation significantly reduces this time frame to less than threemonths.

2. Screening Biological Molecules for Angiogenesis Activity

The present invention also includes methods of screening of largermolecules, including biological molecules, for an ability to enhance orinhibit angiogenesis activity. These methods comprise administering thecompound to a teleost and detecting a response indicating an enhancementor inhibition in angiogenesis activity. No precise method for screeninglarge biological molecules for angiogenesis activity currently exists.Thus, the methods of the invention are thus of particular value and usein evaluating the use of biological compounds as therapeutics and/orprophylactics for treating a variety of diseases in humans associatedwith angiogenesis processes, including neurological diseases,cardiopulmonary diseases, ischemia, developmental diseases, autoimmunediseases, diseases of bone and cartilage, and cancer.

A wide range of biological compounds, including peptides, proteins,glycoproteins, nucleic acids (e.g., DNA and RNA), lipids, glycolipids,and the like, including, but not limited to, derivatives, analogues, andchimeras of such compounds, can be screened by these methods. Asdiscussed above, compounds from a library of compounds, including acombinatorial library, can be screened.

Recently, a number of biological molecules have been identified thathave either anti-angiogenic or angiogenic effects (Hanahan, Science277(5322):48-50 (1997); Zetter, supra). Some biological compounds havebeen characterized and analyzed for angiogenesis activity in cellcultures and in mice; a few such compounds have been tested intherapeutic and/or prophylactic treatment programs in humans. Comparisonof the results using these compounds and the teleost model and screeningmethods of the present invention would allow a determination as towhether the teleost model and screening methods described herein ispredictive of the therapeutic effect in humans; such a comparison wouldbe of benefit in determining whether a tested compound would be usefulin programs for therapeutic and/or prophylactic treatment ofangiogenesis-related disorders in humans.

A. Proteins

To examine the effects of proteins and protein fragments (and peptidesand peptide fragments) on angiogenesis in teleost embryos, proteins (andprotein fragments and peptide fragments) were directly injected into thecirculation of 24 hour zebrafish embryos. Embryos were collected anddechorionated as described above. The embryos were then sorted intoholding ramps made of 1% agarose in embryo water and oriented with theyolk ball projecting up. Microinjection injection was performed asfollows: the proteins were suspended in PBS and backfilled into a pulledglass micropipet. The micropipet was then attached to a micromanipulatorand a picospritzer (General Valve) attached to a nitrogen tank. Usingthe micromanipulator, the tip of the micropipet was inserted into theembryo and a small volume of protein solution was expelled from the tipusing positive pressure. To determine if our animal model could be usedeffectively to screen for these compounds, we performed a series ofexperiments in which we injected one of two different proteins havingopposing effects on vessel formation—human endostatin (O'Reilly et al.,Cell 88(2):277-285 (1997)) and human vascular endothelial growth factor(VEGF)— into an embryo. Endostatin, a collagen XVIII fragment, is anendogenous protein with potent anti-angiogenic activity. VEGF has beenshown to play a critical role in both endothelial cell determination, aswell as vessel formation. In preliminary experiments, we injected VEGFprotein either into the yolk ball or into the perivitelline spacebetween the yolk and the periderm. Because the second location is in thepath of the venous return, proteins end up in the circulation of theembryo. To backfill the injection pipettes, we used a 2 mg/ml solutionof VEGF. When VEGF was injected into the yolk, we observed twoangiogenic phenotypes: 1) the appearance of long spikes projecting fromthe subintestinal vessel basket (FIG. 6A); and 2) increased vesseldiameters in the subintestinal basket (FIG. 6C). In contrast, injectionsof VEGF into the perivetellin space led to a disruption of vesselformation (FIG. 6D) and heart development. This is consistent withobservations in other vertebrates. Drake et al., Proc. Natl. Acad. Sci.USA 92(17):7657-7661 (1995); Fouquet et al., supra. Endostatin wasinjected into the zebrafish as was VEGF. In contrast with VEGF,endostatin results ere inconsistent and thus uninterpretable. Theseexperiments demonstrated that changes in the vascular pattern can beinduced in our animal model. Moreover, because human proteins producedthese effects, these experiments suggested that the mechanisms forangiogenesis in zebrafish and humans are probably similar.

B. Nucleic Acids

To deliver nucleic acids to teleosts, we established a microinjectionsystem. Microinjection of DNA, RNA, and proteins is a well establishedprocedure used in a variety of biological systems, including singlecells, frog embryos, mouse embryos, and zebrafish. Westerfield, supra.In the zebrafish, it is possible to load every cell of the embryo byinjecting molecules of interest into the yolk of 1-16 cell stageembryos. See Westerfield, supra. Using these standard approaches,several hundred embryos can be loaded in a two-hour period.

3. Evaluation of Biolistic Cell Loading Technology

Biolistic cell loading technology uses coated particles to introducemolecules of interest into tissues and organs of an animal. In thistechnique, particles coated with the biological molecule are“biolistically” shot into the cell or tissue of interest of the animalusing a high-pressure gun. This technique has been used successfully toload primary culture cells as well as whole mouse embryos with large DNAplasmid constructs. Chow et al., Amer. J Pathol. 2(6):1667-1679 (1998).

With the methods of the invention, biolistic cell loading can be used asan alternative to microinjection techniques to inject compounds intoanimals, such as, e.g., adult, larval, and teleost embryos. DNA can beregionally administered to the teleost (e.g., introduced to specificlocations within the teleost embryo), such as the tail or the dorsalsurface of the yolk, prior to, after, or at the time angiogenesisbegins.

4. Establishing Parameters for Quantifying and Characterizing theEffects of Compounds on Angiogenesis Activity and Endothelial CellToxicity

To determine whether a particular compound is of potential therapeuticor prophylactic use, a number of additional parameters, including theTherapeutic Window and the Effective Window can be determined.

A. Therapeutic Window

The Therapeutic Window (TW) is the ratio of the Median EffectiveConcentration (EC50) to the Median Lethal Concentration (LC50) (i.e.,LC50/EC50). LC50 is determined by administering serial dilutions of anagent and determining what proportion of teleosts die at each dilution.LC50 is the concentration needed to cause lethality in 50% of theteleosts. Agents which exhibit a high Therapeutic Window (LC50/EC50),such as 100 or 1,000, are good potential drug candidates becausetoxicity at the therapeutic concentration is low. Agent concentrationstypically range from picomolar to millimolar.

B. Effective Window

The Effective Window (EW) identifies the point during angiogenesis atwhich a compound is effective. This is determined by exposing embryos tothe EC50 of a compound at different stages of angiogenesis, beginningwith the 12 somite stage, when angioblasts are first detectable, throughthe 72 hour stage, when vascularization in the embryo is complete.

In our preliminary studies, we identified a number of compounds whichwere toxic at various concentrations. It is possible that such compoundsare extremely potent and that only low (picomolar) concentrations ofsuch compounds effect angiogenesis. This problem can be addressed byscreening compounds for angiogenic effects at concentrations well belowthe concentration at which induces toxicity.

C. Quantitation of Vessel Growth

While visual comparison of an embryo treated with a compound of interestwith an untreated embryo (control) is an effective means for identifyingchanges in the vessel architecture related to angiogenesis, it does notpermit quantitative assessment. As an alternative or in addition tovisual comparison, image analysis can be used to quantify andstandardize the analysis. A number of commercially available softwarepackages exist (e.g., Image-Pro Plus™, Media Cybernetics; WSR ImageAnalysis System, WindSword Software Research; MetaMorph®, UniversalImaging Corp.) that permit both distance and area measurements of vesseldimensions and distribution—the parameters used for visual analysis.

D. Evaluation of Additional Markers for Characterizing AngiogenicActivity

Antibody markers that label signaling proteins involved in angiogenicvessel formation in the zebrafish, including VEGF and Ang1 and 2(Hanahan, Science 277(5322):48-50 (1997)), would assist in identifyingcompounds that are either agonists or antagonists of the signalingmolecules that guide vessel development and patterning. A number ofantibody markers have been identified in mouse and are commerciallyavailable (Santa Cruz Biotechnology, Inc.). These markers can be testedin teleosts using standard antibody staining protocols. Westerfield,supra. Antibodies can be used in place of RNA probes to simplify theassay procedure.

Briefly, embryos are fixed for 2 hours at room temperature. The embryosare then washed two times in phosphate buffered saline with Tween (PBT)and permeabilized by treatment acetone at −20° C. for 7 minutes. Theembryos are rehydrated and then treated with a blocking solution (2%goat serum, 1% bovine serum albumin (BSA) in PBT) for 30 minutes at roomtemperature. Next, the embryos are soaked in blocking solutioncontaining the primary antibody overnight at 4° C. The embryos are thenbe washed 5 times in PBT with 1% BSA. The embryos are soaked in blockingsolution containing a secondary HRP-conjugated antibody for 4 hours atroom temperature. The embryos are then washed and stained by soaking inDAB solution (1 mg diaminobenzidine, 1 ml 0.1M PO₄ buffer, 1 ml dH₂O and20 μl of DMSO) for 15 minutes. H₂O₂ is then added to the solution forcolor development. The reaction is stopped by adding PBT.

E. Assays for Compounds that Induce Endothelial Cell Toxicity

Although the strategy of blocking new vessel formation has significantpotential for anticancer therapeutics, an alternative strategy is todestroy vessels already present in the tumor. With such a strategy, acompound is administered to the teleost after vessel formation, notprior to vessel formation. It is not known how long compounds persist inthe media containing teleost embryos; we assume that effects on bloodvessel formation occur relatively soon after administration of thecompound. To identify compounds that have toxic effects on blood vesselsafter formation, we administered compounds to zebrafish embryos at 60hours of development, when the subintestinal vessels were wellestablished. We then assay the embryos at 72 and 84 hours ofdevelopment. Compounds were screened for those which caused a loss ofthe subintestinal vessel staining, as described above.

F. Evaluating the Use of Mutant Fish Lines

Studies suggest that it may be difficult to induce additional vesselgrowth in a normal animal system. For example, there is evidence of thisin the mouse model for ischemia (Couffinhal et al., Amer. J Pathol.2(6):1667-1679 (1998)). This issue can be circumvented by performingscreens on animals in which vessel development has been impaired. A fewgenetic mutations exist in the zebrafish that disrupt vessel formation.Examples of such mutations are: 1) gridlock, alocalized heritablevascular patterning defect in the zebrafish (Weinstein et al.,Cardiovascular Research Center, Massachusetts General Hospital,Charleston, Mass. (1998)), in which vessel formation is normal in thehead region and absent in the tail for the first 3-4 days ofdevelopment, and after ˜4 days, collateral vessels begin to appear inthe tail; 2) cloche (Fouquet et al., supra, Thompson et al., Dev. Biol.197(2):248-49 (1998)), in which angioblast development is impaired; and3) no tail and floating head (Fouquet et al., supra), notocord mutants,in which the formation of the large vessels is blocked. The usefulnessof these and other mutant lines are readily evaluated by using themethods for screening compounds for angiogenic activity describedherein. Currently, there are no known mutations that cause increases invessel growth in zebrafish.

G. Assessing Effects of Angiogenic/Anti-Angiogenic Compounds on OrganSystems

The effects of a compound on organ systems other than the vascularsystem (e.g., kidney, heart, etc.) can be determined by using screeningmethods described herein. The ability to make such determinations issignificant, because in evaluating the potential therapeutic value ofany compound identified using the methods of screening compounds forangiogenesis activity, it is important also to identify adverse effects,including adverse effects on other organ systems. The teleost model isideal for this purpose because many of its organs can be visualized inthe transparent teleost embryo by light microscopy (e.g., the heart andthe CNS); alternatively, a number of organs of the teleost embryo can beidentified by simple staining techniques (e.g., liver, gut, heart, andkidney). For example, cardiac function and liver viability can beassayed. Because the heart is both directly connected to the vasculatureand because the heart and the vessels share some of the same cell types,the heart is a likely secondary target of compounds that affectangiogenesis activity. Because the liver is the site of accumulation andmetabolism of many compounds, especially toxins, it is an indicator ofthe toxicity of both the compounds and the breakdown products.

As described above, in our initial studies, we observed that a smallnumber of compounds affected the heart rate of the zebrafish embryo.With six compounds, we observed that the zebrafish heart beat atapproximately 1-2 beat(s) per second instead of the normal 4-5 beats persecond. To determine if a particular compound affected teleost heartdevelopment, or if it acted as an antagonist to the conductivitymechanism, we administered the compound of interest to a zebrafishembryo at 72 hours of development, when a functioning heart and vascularsystem is present. The embryos were then evaluated 2 hours after theaddition of the compound for immediate effects on heart rate andcontractility and at 24 hours for effects which might require novel geneor protein expression. If a compound acted as a direct antagonist oneither the conduction or contraction machinery of the zebrafish heart,its administration to the zebrafish at any stage would likely show aneffect. However, if a compound affected development of the zebrafishheart, its presence should show no effect at the later stage ofdevelopment.

In addition to examining the heart rate and contractility, we alsoexamined the structure of the heart both by visual inspection (Stainieret al., Development 123:285-92 (1996)) and by staining the heart withantibodies against tropomyosin and cardiac myosin heavy chain (Stainierand Fishman, Trends Cardiovasc. Med. 4:207-212 (1994), which allowedidentification of the atrium and the ventricle—the two chambers of thefish heart. Briefly, embryos were fixed for 2 hours at room temperature.The embryos were then washed two times in PBT with Tween andpermeabilized by treatment acetone at −20° C. for 7 minutes. The embryoswere rehydrated and then treated with a blocking solution (2% goatserum, 1% bovine serum albumin (BSA) in PBT) for 30 minutes at roomtemperature. Next, the embryos were soaked in blocking solutioncontaining the primary antibody overnight at 4° C. The embryos were thenwashed 5 times in PBT with 1% BSA. The embryos were subsequently exposedto the appropriate fluorescent conjugated secondary antibodies fordetection. The embryos were analyzed using an epifluorescencemicroscope. This method uses a particular means of detection;alternative secondary reagents and visualization (or detection) methods,including, e.g., chromogenic, radiographic or other methods, may beused.

III. Methods of Screening an Agent for an Effect on Cell Death Activity

A. Cell Death

The death of cells of multicellular organisms may result from naturalprocesses or external non-physiological causes. Two types of cell deathare known: necrosis and apoptosis. Necrosis is the pathologic death ofliving cells which results from acute, non-physiological injury to thecells. Hetts, J. Amer. Med. Assoc. 279(4):300-07 (1998). Necrosis mayresult from the exposure of a cell to a number of differing conditions,including toxins, severe hypoxia, massive insult or physical injury, orconditions of adenosine 5′-triphosphate (ATP) depletion. Id. Necrosisoccurs, for example, in the center of infarcted tissue in an ischemicstroke or at the center of toxin action. Id. Necrotic cells swell andlyse, thereby releasing their nuclear contents into the surroundingintercellular regions and causing an inflammatory response. Id.Significantly, however, necrosis is not the only mechanism by whichcells die.

Apoptosis, or programmed cell death, is a naturally-occurringphysiological process that plays an important role in modeling tissuesduring development. Kerr et al., Br. J. Cancer 26:239-257 (1972);Clarke, Anat. Embryol. 181:195-213 (1990). Apoptosis ensures that abalance is maintained between cell proliferation and celldifferentiation in nearly all self-renewing tissues of multicellularorganisms. Apoptosis allows the elimination of cells that are, forexample, no longer required, are produced in excess, have incurreddamage, or have developed improperly. Numerous types of cells undergocell death through by apoptotic processes. Hetts, J. Amer. Med Assoc.279(4):300-307 (1998). Apoptotic cells undergo a number ofcharacteristic changes, including chromatin condensation, nuclearfragmentation and cytoplasmic blebbing. Liepins and Bustamante, ScanningMicrosc. 8:631-641 (1994). This programmed cell death mechanism isprecise and predictable, and the stages and genes that govern cell deathare highly conserved among multicellular animals.

Apoptosis appears to be directed by the dying cells themselves, andduring development, it is involved in maintaining the appropriate cellnumber and cell type in a given organ or tissue. Some apoptotic eventsare believed to be regulated by limiting the amount of growth orsurvival factors. It can also be triggered in response to externalstimuli, including, for example, radiation, hyperthermia, hormonewithdrawal, immune reactions, radiation, chemotoxins, temperatureextremes, growth factor deprivation, and infection by some viruses.Thompson, Science 267:1456-1462 (1995).

Abnormal regulation of apoptosis has been implicated in the onset andprogression of a broad range of diseases resulting from inappropriatecell death or inhibition of cell death. Apoptotic dysregulation has beenimplicated, for example, in some types of cancer cells which survive forlonger periods than do corresponding normal cells. It is believed thatthe suppression or failure of the apoptotic mechanism allows certaincancer cells to undergo further mutations leading to a transformed orcancerous state. Hetts, J. Amer. Med. Assoc. 279(4):300-307 (1998).Uncontrolled apoptosis has also been implicated in other disorders,including neurodegenerative disorders, lymphoproliferation, autoimmunediseases, and heart and renal diseases. Id.

In addition, many therapeutic approaches for diseases (e.g., cancer,heart disease, and neurodegenerative diseases), including variouschemotherapies and organ transplantation, have been shown to induceapoptosis in normal cells. Id.

Indiscriminate inhibition of apoptosis can lead to widespreadhyperplasia, and inappropriate promotion of apoptosis may lead toundesirable tissue degeneration, underscoring the need for more preciseassays for studying cell death. Multiple pathways to apoptosis mean thatdifferent therapeutic approaches are possible for treating abnormalapoptotic regulation, demonstrating the need for assays for screeningcompounds for their ability to cause or suppress cell death.

Understanding of the mechanisms of cell death activity, includingapoptosis, would facilitate the development of therapeutic compoundsthat either stimulate, trigger, or initiate cell death, or,alternatively, suppress, inhibit, or block cell death. For example, thediscovery of signaling proteins and their corresponding receptorspresents the opportunity for the development of tools for correcting theapoptotic cellular machinery when it goes awry or harnessing itspotential for cell killing. Because abnormal regulation of apoptosis hasbeen implicated in the onset and progression of a wide range ofdiseases, many disorders can now be classified based as to whether theyare associated with too much or too little apoptosis. In particular, anumber of approaches aimed at cancer therapy are currently underinvestigation, since it is known that tumor cells proliferate when theapoptotic engine fails to operate. Potential methods of repair includefinding chemicals that target receptors to restore the apoptoticfunction in tumor cells, and inducing apoptosis in a tumor's developingblood vessel.

Improved understanding of the molecular apoptosis pathways may alsostimulate development of novel non-pharmaceutical therapies. Forexample, an adenovirus that is only able to replicate in and killp53-deficient cells is currently in Phase I clinical trials as apossible antitumor agent that should kill only p53 deficient tumorcells, leaving normal cells unaffected. A compound that limits coronarydamage if injected after a heart attack is also presently underinvestigation, underscoring the potential for the development ofmolecular approaches utilizing small molecules that both inhibit andinduce apoptosis selectively. Improved understanding of thephysiological process of apoptosis at the molecular level would provideinsight into disease pathogenesis and open new avenues for developingdiagnostic, prognostic, and therapeutic tools.

The genetics and molecular mechanisms of apoptosis were characterized inthe late 1980s and early 1990s in studies using the nematode worm, C.elegans. Although the nematode has many advantages as a model system,including evolutionary conservation of much of the signaling pathwayinvolved in apoptosis (see, e.g., Steller, Science 267:1445-49 (1995)),it is not the optimum model for an understanding of vertebrate celldeath activity and disease states. Vertebrates are much more complex andhave multiple apoptosis pathways comprised of many more signalingmolecules. There are currently no rapid in vivo assays of screening acompound for its effect on cell death activity, such as apoptosis, invivo in a vertebrate system. It would therefore be desirable to providea rapid in vivo method of screening a compound for its effects on celldeath activity, including apoptosis and necrosis, in a vertebratesystem.

Currently, there are two primary approaches for detecting cell deathactivity in vertebrates hosts. The first approach uses standard cellsculture techniques and typically relies on standard microplate platereaders to detect the death of cells cultured from an organism. A majordrawback of the cell culture assay format is that it does not permitanalysis of the effects of a compound on cell types that have not beencultured (i.e., other cell types) or on one or more tissues or organs oran intact, whole host in vivo. Furthermore, such an assay format doesnot permit simultaneous monitoring of cell death activities in multipletissues, organs, or systems of a live host or monitoring over time.

A second approach to detecting cell death activity utilizes ahistochemical staining technique, designated terminal deoxyuridinenucleotide end labeling (TUNEL) to detect dead or dying cells (e.g.,apoptotic cells) in sectioned tissues of vertebrate embryos. Gavrieli etal., J. Cell. Biol. 119:493-501 (1992). Unfortunately, with thisapproach, only a single time point in the life cycle of the host can beexamined; the death of cells in various tissues or organs over a periodof time cannot be monitored. Nor can side effects due to an administeredcompound be monitored simultaneously or over time. Because many diseasesoccur in stages, the ability to examine changes in the pattern of celldeath activity caused by a compound, the duration of direct and sideeffects of the compound of multiple tissues, would represent asignificant improvement over current methods.

Gene products that regulate cell death activity, including apoptosis,are excellent targets for therapeutic intervention in alleviating manydisease processes. Few such therapeutic gene products currently exist.It would be also desirable to provide a method of screening a compoundfor its potential therapeutic effect on cell death activity. Suchmethods would be of benefit in alleviating diseases resulting fromabnormal cell death processes, including those resulting frominappropriate cell death or inhibition of cell death (e.g., apoptoticdysregulation).

B. Methods of Screening Agents for Cell Death Activity

The present invention provides methods of screening an agent for aneffect on cell death activity in a vertebrate animal, such as a teleost,in vivo or in vitro in cells of the animal. Cell death activity is theability or capacity of an agent to enhance, stimulate, inhibit, or blockcell death in an animal, tissue, organ, or cell in response toadministration of an agent. Cell death activity is assessed relative tocontemporaneous and/or historical control teleosts (or tissues, organs,or cells thereof) to which the agent has not been administered. Suchmethods are useful for screening an agent for its ability to trigger,enhance, suppress, or eliminate apoptotic or necrotic processes.Identified agents can be used potentially in therapeutic or prophylactictreatment of diseases which result from abnormal cell death processes ordiseases which would benefit from the elimination or controlled death oftargeted cells or tissues.

Some such methods comprise administering the agent to a whole teleost invivo and detecting a response in the teleost indicating an effect oncell death activity. Other such methods comprise administering the agentin vitro to a culture of cells of a teleost and detecting a response inthe cells indicating an effect on cell death activity. In some suchmethods, the detected response is an increase or initiation of celldeath activity. In other methods, the detected response is a decrease orsuppression of cell death activity. In some methods, the response is anincrease or decrease in apoptotic activity. An effect on apoptoticactivity can be measured by detecting a response indicating such aneffect; the response can be, for example, an increase or triggering ofapoptosis or a decrease or suppression of apoptosis. An increase inapoptotic activity generally comprises an increase in the death of cellsin a tissue or organ of the animal.

Typically, the animal is a teleost, such as a zebrafish. Usually, theteleost is transparent. The teleost can be in embryonic, larval, oradult form.

Alternatively, an agent can be screened for an effect on necroticactivity in vivo in a teleost by administering the agent to the teleostin vivo and detecting a response in the teleost indicating an effect onnecrotic activity. An agent can also be screened for an effect onnecrotic activity in vitro by administering the agent in vitro tocultured cells of a teleost and detecting in the cells indicating aneffect on necrotic activity. In both such methods, the response can bean increase or decrease in necrotic activity.

The effect of a particular agent on the entire, intact teleost and/orone or more organs, tissues, or systems of the teleost (e.g., thecardiovascular system, the enteric system, and the musculature) can bemeasured in vivo or in vitro in cells of the teleost and, if desired,over a period of time and/or at selected time intervals. Responses incombinations of organs and/or tissues can be detected simultaneously orseparately; such analyses can be performed over time at predeterminedtime intervals. These methods can also be used with isolated cells orcell lysates.

Cell death activity can be detected in vivo or in vitro by using atleast one of a variety of techniques, including, e.g., fluorescencemicroscopy, light microscopy, digital image analyzing, or standardmicroplate reader techniques (colorimetry, fluorometry, includingtime-resolved fluorometry, and chemiluminescence), antibody staining ofproteins, changes in enzyme levels or enzymatic activities in the wholeteleost, or tissues, organs or cells of the teleost, and changes inprotein distribution temporally and spatially within the animal. Theresponse can also be discriminated and/or analyzed by using patternrecognition software. Thus, for example, an increase in apoptotic ornecrotic tissue can be analyzed in a zebrafish by using such techniques.

Fluorescence-based detection techniques and fluorescence microscopy canalso be used to detect the effect of an agent on cell death activity inan animal, such as a teleost. For example, teleosts can be stained witha membrane-impermeant, nuclear-staining fluorescent dye which permitsdetection of cell death activity (e.g., apoptosis or necrosis). Avariety of fluorescent dyes can be used. Preferred dyes include those ofthe unsymmetrical cyanine dye family (such as quinolium dyes, e.g.,benzothiazolium-4-quinolium dyes (Molecular Probes)), includingderivatives, analogs, and substituted or unsubstituted forms thereof.Such dyes are generally discussed in U.S. Pat. No. 5,658,751, which isincorporated herein by reference in its entirety for all purposes. Anumber of these dyes are commercially available.

These dyes, including monomeric cyanine dyes (such asbenzothiazolium-4-quinolium), cannot pass through intact membranes ofcells of live embryos. However, these dyes can enter dead or dying cellswhose membranes have become discontinuous or disrupted (a characteristicof cells undergoing cell death, Liepins and Bustamante, ScanningMicrosc. 8:631-41 (1994)). Notably, the cytoplasmic blebbing and otherproperties in the membrane characteristic of a dead, dying cell, orapoptotic cell permit such dyes to enter the cell.

Upon passing through the cell membrane, monomeric cyanine dyes (e.g.,benzothiazolium-4-quinolium) intercalate into the DNA of the dead ordying cells. The dense chromatin and nuclear fragmentation provide anideal environment for dye intercalation and signal amplification.Singer, Biotechnol. Intl. 1:267-276 (1997). Upon intercalating into theDNA, the dye becomes intensely fluorescent, allowing for rapid detectionof the labeled cells using simple fluorescent microscopy. Notably, whenconcentrated in DNA, the fluorescent signal ofbenzothiazolium-4-quinolium dye is amplified up to 400 fold. Serbedzijaet al., J. Neurobiol. 31(3):275-282 (1996). The magnitude of the signalserves as a measure of the number of apoptotic or necrotic cells.

Notably, the in vivo methods of screening agents for cell death activityof the present invention provide a more sensitive and accurate detectionand measurement of cell death in whole embryos in vivo than permitted byexisting approaches. Other fluorescent markers of cell death, such asAcridine Orange, pass through the membranes of cells of live embryosmuch more readily and fluoresce under a variety of conditions than domonomeric cyanine dyes, such as benzothiazolium-4-quinolium dyes. Forexample, Acridine Orange fluoresces when bound to nucleic acids and whenlocalized in subcellular compartments such as lysozymes. It has alsobeen reported that Acridine Orange does not bind effectively with DNAunder some circumstances, including chromatin compaction which issometimes associated with apoptosis. Thus, Acridine Orange and similarsuch dyes do not provide as specific a marker of cell death as abenzothiazolium-4-quinolium dye.

Monomeric cyanine dyes (e.g., benzothiazolium-4-quinolium dyes) alsoprovide a higher signal-to-background when bound to nucleic acid than doother fluorescent markers of cell death, such as Acridine Orange. Inaddition, the fluorescence emission spectra ofbenzothiazolium-4-quinolium dyes are typically narrower (i.e., emissionoccurs over a narrow wavelength emission band) than are the emissionspectra of other fluorescence labels, such as, e.g., Acridine Orange,which has a very broad fluorescence emission spectrum. Thecharacteristic emission spectra of monomeric cyanine dyes permit the useof two or more additional fluorescence labels simultaneously inconjunction with the quinolium dye, thereby allowing characterization ofmultiple types of physiological events within the same or differentorgans or tissues. A broad emission spectrum (e.g., Acridine Orange)severely limits the ability to use multiple fluorescent labels forscreening methods described herein due to overlap between thefluorescence emission spectra of the labels. Thus, with the methods ofthe invention described herein, more than one fluorescent dye can beused together for monitoring multiple cellular and/or molecularphenomena in response to an agent administered to the animal in vivosimultaneously over time. Dyes can be selected to have emission bandsthat match commercially available filter sets such as that forfluorescein or for detecting multiple fluorophores with severalexcitation and emission bands.

Furthermore, in contrast with other fluorescent markers of cell death,benzothiazolium-4-quinolium dyes are not toxic; thus, apoptotic ornecrotic effects in a living teleost to which the dye has beenadministered can be monitored over a significant time period, withoutrisk that the teleost will be adversely affected by the dye. Incontrast, assays using other types of markers of cell death require thatthe host be sacrificed and fixed (e.g., TUNEL labeling).

The fluorescent dye is typically administered to the teleost by addingthe dye to the media containing the teleost. Alternatively, the dye canbe injected directly into the teleost. The dye is typically administeredprior to administration of the agent to be screened for cell deathactivity. This procedure provides superior results over existingapproaches, because we have found that if the dye is added afterapoptosis has been induced, the dye is less effective in labeling deador dying cells. One of the apoptotic mechanisms (e.g., thepolymerization of components of intracellular membranes and the plasmamembrane) may make it difficult or impossible for the dye to enter thecell. As a result, an apoptosing cell may not be labeled. By applyingthe dye prior to application of the agent, this problem is avoided. Thefluorescence emission of the dyes is monitored by using standardfluorometric techniques, including visual inspection, CCD cameras, videocameras, photographic film, or the use of current instrumentation, suchas laser scanning devices, fluorometers, photodiodes, quantum counters,photon counters, plate readers, epifluorescence microscopes, scanningmicroscopes, confocal microscopes, or by means for amplifying thesignal, such as a photomultiplier tube.

Unlike other known dyes which involve laborious labeling procedures(e.g., TUNEL labeling), the benzothiazolium-4-quinolium dyes areparticularly suitable for high throughput, automated screening methods.The higher signal-to-noise ratio inherent in these dyes and our superiormethod of administering the dye prior to administration of the agent tobe screened for apoptosis enable automated data acquisition, moreaccurate quantitation of the collected data (e.g., digital imaging), andthe possibility of feature extraction/image segmentation of acquireddata. These features allow mapping of the apoptosis signal in space/timedimensions that can be correlated with fate map coordinates of thespecific teleost's fate map. Such information permits furthercharacterization of the screened agents.

As noted above, cell death activity (e.g., apoptosis and necrosis) canalso be detected by digital imaging. Digital imaging is an indispensabletool for biological research due to several advantages when compared tothe human eye. Digital imaging involves the collection of images using acharge-coupled device (CCD). The higher sensitivity imaging detectorenables one to visualize very low light objects which are not detectableby the unaided human eye. The spectrum sensitivity of the human eye islimited from 400 to 700 nm. In contrast, the spectrum sensitivity rangeof imaging detectors is more broad, and signals from the range of x-rayto infrared can be detected. Combining digital mapping and patternrecognition software enables the quantification and comparison ofmultiple data sets and facilitates comparison of contemporaneous andhistorical controls with experimental teleost animals.

The present invention also provides methods of screening a compound forits effect on cell death activity in vivo in a teleost or in vitro incells of the teleost over time. Such methods comprise administering thecompound to the teleost in vivo or in vitro in cells thereof, detectinga response in the teleost indicating an effect on cell death activity,and further detecting a response in cell death activity in the teleostafter a predetermined period of time or time interval. The period oftime, which is selected by the practitioner, is typically sufficient fordetectable cell death to occur in the presence of the compound. Inaddition, multiple time points can be examined to detect any pertinentphysiological activity. Some such methods further comprise detecting aresponse in cell death activity (e.g., apoptosis) after a secondpredetermined time interval using the detection techniques describedherein. Such methods are useful in evaluating the effect of an agent(e.g., chemical compound, drug, environmental agent, agriculturalcompound, toxin, pharmaceutical, cosmeceutical, etc.) on tissues andorgans over time in the intact, live teleost.

In yet another aspect, the present invention provides methods ofscreening an agent for an effect on cell death activity in vivo or invitro, as described above, which further comprise detecting an increaseor decrease in cell death activity in more than one tissue or organ ofthe teleost simultaneously. In some such methods, the increase ordecrease in cell death activity is detected simultaneously in more thanone tissue or organ at predetermined time intervals. The effect of aparticular compound on various cells, tissues, and organs of the embryocan be monitored and assessed over time. Cell death activity in multipletissues or organs can be detected by using the detection techniquesdescribed throughout this specification.

The present invention also provides automated methods of screening acompound for an effect on cell death activity in vivo or in vitro. Themethods of the invention can be performed using a standard microplatewell format, with one or more whole teleosts per well of the microplate.This format permits screening assays to be automated using standardmicroplate procedures and plate readers to detect cell death in thezebrafish in the wells. With this setup, the effect of a specificcompound on a large number of teleosts can be ascertained rapidly. Inaddition, with such format, a wide variety of compounds can be rapidlyand efficiently screened for their respective effects on the cells ofteleosts (e.g., teleost embryos) contained in the wells. Both samplehandling and detection procedures can be automated using commerciallyavailable instrumentation and software systems for rapid reproducibleapplication of dyes and compounds and automated screening of targetcompounds.

The contemporaneous and/or historical control teleosts (which includesteleost tissues, organs, or cells) used with these methods can includethose in which at least one inhibitor of apoptotic molecular mechanisms(including, e.g., known or specific inhibitors of apoptotic mechanisms)has been administered to the teleost (or tissues, organs, or cellsthereof) at specific stages of development, thereby generating aparticular phenotype, such as tissue malformation (e.g., expansion ofthe central nervous system; malformation of the cloacal/anal poreregion; hyperproliferation of cells in any tissues). Agents can then bescreened for the ability to induce the same or similar phenotype (i.e.,phenotypic response). With such methods, the agent is administered tothe teleost as described above; the phenotypic response in the teleostcan be detected visually by light microscopy, by fluorescent labelingwith unsymmetrical cyanine dyes discussed above, or by labeling with insitu hybridization or antibody staining for selected cell types. Usingthese types of controls, the agents can be screened for the ability to“phenocopy” the effect of the loss of molecular function(s) ormechanism(s) induced by the apoptotic inhibitor. Phenocopying in theexperimental teleost (or tissues, organs, or cells thereof) relates tothe duplication or mimicking of the same or similar phenotype observedin the control.

The present invention includes screening methods which rely on detectingenzymatic activity associated with apoptosis. In one aspect, theinvention provides methods of screening an agent for apoptotic activitywhich comprise administering the agent to a teleost and detecting aresponse in the teleost indicating apoptotic activity by detecting theactivity of an enzyme (e.g., cleavage of caspase substrate).

Caspase enzymes, for example, are well characterized proteases thatfunction as triggers, effectors, or mediators in a number of apoptoticpathways. The fluorogenic caspase substrate can be introduced into theteleost by a variety of methods, including, e.g., by injection into theteleost, by dissolving the substrate in the medium containing theteleost. The manner of introduction of the substrate depends upon theparticular type and nature of reporter substrate design (e.g., smallmolecule, plasmid). The fluorogenic caspase substrate can be introducedat the time or, after, or, usually, prior to administration of theagent. Caspase activity (e.g., cleavage of caspase substrate) can bemeasured by using, for example, commercially available colorimetric orfluorometric enzymatic assays or by using antibodies which detectcleaved substrates (e.g., M30 CytoDEATH antibody; Boehringer Mannheim).Specific patterns of embryo dysgenesis result from the inhibition ofnaturally occurring apoptotic events during development. Inhibition ofcaspase activity can cause specific morphological effects includingtissue malformation. Such methods can be conducted in vivo using wholeteleosts or in vitro using cells of the teleost. Such methods are usefulfor identifying agents having apoptotic activity that may have potentialtherapeutic or prophylactic use for treating a variety of diseases, suchas cancer.

C. Screening Agents for Cell Death Activity and/or Angiogenesis Activityand/or Toxic Activity Simultaneously

The methods for screening agents for cell death activity can be combinedwith other methods of the present invention, including methods ofscreening agents for angiogenesis activity (Section II) or toxicactivity (Section IV). Because teleosts are transparent, it is possibleto assess effects of cell death activity, angiogenesis activity, and/ortoxic effects in teleosts in response to an agent simultaneously.Responses can be monitored in one or more tissues or organs and atpredetermined time intervals.

As noted previously, these combined methods are useful in assessingmultiple effects of an agent, including desired and undesired responses(such as detrimental side effects) and dose levels of the agenteffective to promote one activity without promoting the other. Theability to assess multiple activities and responses in an animal due tothe administration of an agent is of particular benefit in identifyingpotential therapeutic compounds and assessing their side effects.Pathological regulation of apoptosis, for example, is associated with awide variety of human diseases including cancer, heart disease,neurodegenerative disorders, and immune, renal and viral-induceddiseases. Essentially all cells are poised to commit suicide from theearliest stages of development. Thus, it is imperative that drugs beexactingly targeted. A balance must also be achieved during treatmentwith drugs such that only a negligible level of cell death and toxiceffects in non-targeted tissues or organs. The combined methods of theinvention are useful in assessing the specificity and extent of celldeath and deleterious and toxic effects of potential drugs in particularorgans and tissues or within the whole animal.

A variety of techniques can be used together or separately to analyzemultiple activities/responses, including, e.g., fluorescence microscopy,light microscopy, digital image analyzing, standard microplate readertechniques (colorimetry, fluorometry, including time-resolvedfluorometry, and chemiluminescence), radiometric analysis, in situhybridization, changes in enzymatic activity and levels in the wholeteleost, or tissues, organs or cells of the teleost, antibody stainingof specific proteins, changes in protein distribution temporally andspatially within the animal, etc.

In one aspect, the present invention provides methods of screening anagent for an effect on cell death activity in vivo or in vitro asdescribed above which further comprise screening the agent for anincrease or decrease in toxic activity by detecting a response in theteleost indicating an increase or decrease in toxic activity. In anotheraspect, the invention provides methods of screening an agent for aneffect on cell death activity in vivo or in vitro as described abovewhich further comprise screening the agent for an increase or decreasein angiogenesis activity by detecting a response in the teleostindicating an increase or decrease in angiogenesis activity.

With such combination methods, the contemporaneous and/or historicalcontrol teleosts (or tissues, organs, or cells thereof) for the celldeath activity screens can include those described above in which atleast one inhibitor of apoptotic molecular mechanisms has beenadministered to the teleost (or tissues, organs, or cells thereof) atspecific stages of development, thus generating a particular phenotype.Agents can then be screened for the ability to induce the same orsimilar phenotype.

Examples

1. Induction of Cell Death in Zebrafish Embryos

Treatment of zebrafish with retinoic acid (RA, vitamin A acid) leads toincreased apoptosis in a number of developing tissues, including theneural tube. Ellies et al., Mech. Dev. 61:23-36 (1997). Retinoic acid isknown to regulate the expression of the Hox gene family. Members of thisgene family have been shown to confer positional identity to cellsduring development. Hunt et al., Nature 353:861-864 (1991). Retinoicacid induced apoptosis may be a result of a change in a cell's identity,or a direct effect on the apoptosis pathway. In either case, applicationof retinoic acid is different stages of development leads tocharacteristic patterns of cell death (see, e.g., FIGS. 10A-10B).

Normal apoptosis occurs in the zebrafish embryo throughout the completemorphogenesis period of 72 hours; however, high levels of apoptosis canbe induced in the 24-hour embryo by administration of retinoic acid tothe embryo. In methods of the present invention, retinoic acid is usedto generate a reproducible pattern of cell death for assay optimization.Where cell death activity involves apoptosis, assays of the presentinvention can include a comparison of the effects of apoptosis inducedby retinoic acid in zebrafish embryos in vivo (i.e., control) with theeffects of a test compound on zebrafish embryos in vivo. The accuracy ofsuch assays can be confirmed by performing the TUNEL assay (describedabove) on the same zebrafish embryos.

A method of screening a test compound for an effect on cell deathactivity (e.g., apoptosis or necrosis) in vivo in vertebrate embryos isdepicted below:

This method can be readily automated using known automated software andinstrumentation systems.

With this method, zebrafish embryos are first generated by naturalmating and then collected and placed in egg water prepared by combining5 gram (g) of Instant Ocean Salt with 3 g of calcium sulfate in 25liters of distilled water. Embryos are then be treated with a 2 mg/mlpronase solution for 1 minute at 28° C. and washed three times in eggwater to remove the chorions. The embryos are maintained in the eggmedia throughout the experiments. Because the zebrafish embryo receivesnourishment from an attached yolk ball, no additional maintenance isrequired. After 12-14 hours of development, embryos are treated with 0.1μM to 1 μM retinoic acid in 0.5% dimethyl sulfoxide (DMSO) for 2 hoursto induce cell death in the forebrain and hindbrain of the zebrafish.Cell death in the forebrain and hindbrain can be detected atapproximately 24 hours of development (see FIGS. 10A-10B). Embryos arethen be washed twice in egg water and maintained at 28° C. until theyreach the appropriate developmental stage for staining.

2. Fluorescent Detection of Cell Death in Live Zebrafish Embryos

To identify cells undergoing cell death, embryos are stained with amembrane-impermeant, nuclear-staining fluorescent dye, such as a dye ofthe benzothiazolium-4-quinolium dye family. Benzothiazolium-4-quinoliumdyes are soluble in DMSO and can be administered to the zebrafish byadding the dyes directly to the medium, which usually contains DMSO.Zebrafish tolerate high levels of DMSO well.

Zebrafish embryos are generated and the chorions are removed asdescribed above. The embryos are divided into 4 groups:

1) Untreated, unstained embryos (autofluorescence+cell death)

2) Treated, unstained embryos (autofluorescence w/induced cell death)

3) Untreated, stained embryos (fluorescent dye+normal cell death)

4) Treated, stained embryos

Embryos from group 2 and 4 are treated with retinoic acid at 12 hours ofdevelopment as described above. At 20 hours of development, individualembryos from each group are placed in single wells of a multi-wellculture plate (e.g., 96-well culture plate) in 100 microliter (μl) ofegg water. Because zebrafish embryos develop normally in 100 μl ofwater, test compounds and dyes can be easily added directly to themedium in which the fish is maintained. In this procedure, thefluorescent dye is added to the media of embryos from groups 3 and 4 in1/5, 1/10, 1/50, 1/100 and 1/200 dilutions. For each dye concentration,embryos are collected at 30-minute intervals for 4 hours. The embryosare then washed twice in tank water for 5 minutes. The embryos areviewed using an epifluorescence microscope equipped with a CCD camerafor low light level detection. Images are collected and compared usingstandard software (e.g., Photoshop, Adobe System). Group 1 embryosreflect the normal level of autofluorescence. Group 2 embryos reflectautofluorescence caused by the apoptotic cells or retinoic acid. Group 3embryos indicate normally occurring cell death. Group 4 embryos providethe primary baseline for assay development.

To confirm the accuracy of fluorescent detection of cell death (e.g.,apoptosis), a conventional TdT-mediated dUTP-biotin nick end labelingassay (designated terminal deoxyuridine nucleotide end labeling or“TUNEL”) is performed on the same embryos (Groups 1-4, described above).The TUNEL assay is described in Gavrieli et al., J. Cell. Biol.119:493-501 (1992). This assay is a method of in situ labeling of DNAbreaks in nucleic, in tissue sections processed through standardhistopathological procedures. The method employs terminaldeoxynucleotidyl transferase (TdT) to end label DNA fragments within thenucleic of apoptotic cells. TdT specifically binds to the 3′-OH ends ofDNA, ensuring a synthesis of a polydeoxynucleotide polymer. Afterexposure of nuclear DNA on histological sections by proteolytictreatment, TdT is used to incorporate biotinylated deoxyuridine at sitesof DNA breaks. The resulting signal is amplified by avidin-peroxidase,enabling conventional histochemical identification by light microscopy.

Because the zebrafish embryos are transparent, TUNEL staining can bedone in whole mount format. Whole embryos are fixed in 4%paraformaldehyde overnight and stained using the TUNEL proceduredescribed by Gavrieli supra. Embryos are rinsed in ddH20 (doubledistilled water) and 10 mM Tris-HCl, pH 8. Embryos are then pre-treatedwith TdT buffer (30 mM Trizma base, 140 mM sodium cacodylate, pH 7.2, 1M cobalt chloride (COCl₂)) for 3 hours at 37° C. Embryos are then washed3 times for 30 minutes in phosphate-buffered saline with 0.1% Tween(PBST) at pH 7.2. PBST is then replaced with a reaction mixture of TdTbuffer containing 40 μM bio-16-dUTP (Enzo Biochemicals) and 0.3 Units/μlof TdT enzyme (IBI/Kodak) overnight at 37° C. The reaction is terminatedby washing the embryos in 2× saline-sodium citrate buffer (SSC). Embryosare then rinsed in PBS. Biotinylated nucleotides are detected using astreptavidin complex conjugated to horseradish peroxidase (HRP)according to the manufacturer's protocol (A+B reagents, Vectastain). HRPis detected by incubating the sections in a 3,3′-diaminobenzidine (DAB)solution containing 500 μg/ml DAB, 0.2% CoCl₂, 0.2% NiSO₄(NH₄)2SO₄-6H₂Oand 10% H₂O₂ in 1 M phosphate buffer, pH 7.4. Stained embryos arevisualized on a compound light microscope.

As an additional test of our cell death detection method, we alsoexamined the ability of the dye to label cells in embryos treated withIbuprofen, a cyclooxygenase inhibitor, which causes a characteristicpattern of cell death. This pattern consists of a posterior to anteriorprogression of dying cells as indicated by a progression of opacitychanges in the cells of the embryo.

Embryos as 24 hours of development were pretreated for 1 hour with a 100nanomolar concentration of the fluorescent dye (e.g.,benzothiazolium-4-quinolium dye) in embryo media. The embryos were thenexposed to 100 μM of Ibuprofen by addition to the media and examined bylight and epifluorescence microscopy every 15 minutes for 2 hours.Control embryos were pretreated with the same concentration of dye, butwere not exposed to the Ibuprofen. Within 1 hour, fluorescently labeledcells were detected in the posterior tip of the tail in the experimentalembryos, but not in the control embryos. At 1 hour and 30 minutes,labeled cells were detected in a large patch, extended from the tip ofthe tail to the level of the anus. By two hours, fluorescently labeledcells could be seen throughout the embryo, In contrast, no such patternof labeled cells was observed in the control embryos. This pattern offluorescently labeled cells was identical to the pattern observed forthe opacity changes in the embryo.

3. Rescue of Induced Cell Death Activity

To determine if the teleost model could be used to screen for compoundswhich blocked or reduced cell death activity, embryos were microinjectedwith a Caspase 3 inhibitor (Calbiochem # 264155) prior to exposure toIbuprofen.

Specifically, embryos at 24 hours of development were dechorionated andmicroinjected with either 25 μM Caspase 3 inhibitor or PBS into theyolk. At 26 hours of development, embryos were exposed to 100 μMIbuprofen by addition to the media. At one hour, opaque cells wereobserved in embryos injected with PBS, but not in embryos injected withthe Caspase inhibitor. At two hours after the addition of Ibuprofen,embryos injected with PBS were completely opaque. In contrast, embryosinjected with the Caspase inhibitor were still transparent, however,opaque cells had begun to appear in the most posterior region of thetail in these embryos. By 24 hours after the introduction of Ibuprofen,all of the experimental embryos were dead.

4. Screening Compounds for an Effect on Cell Death Activity

A wide variety of compounds can be analyzed for their potential effecton cell death activity (e.g., apoptotic or necrotic activity) by usingmethods of the present invention. Therapeutic or prophylactic drugs,chemicals, toxins, and pharmaceuticals are among those that can betested for their effects on cell death activity, including their abilityto inhibit or trigger apoptosis.

Compounds to be screened can be obtained from various sources, includingthe National Cancer Institute's Natural Product Repository, Bethesda,Md.

A compound to be tested can be administered to a teleost (e.g.,zebrafish) in vivo by dissolving the compound in the solution or mediumcontaining the teleost. The compound is absorbed by the teleost.Alternatively, the compound can be injected directly into the teleost.

When screening compounds for their effects on apoptotic activity, it isuseful to compare an assay utilizing teleost embryos to which the testcompound has been administered with embryos to which retinoic acid hasbeen administered. For such comparative assays, teleost embryos aredivided into four groups:

1) No retinoic acid, no test compound (normal control)

2) Retinoic acid, no test compound (induced cell death control)

3) No retinoic acid, test compound

4) Retinoic acid, test compound

Screening methods are performed as described above. Specifically,zebrafish embryos belonging to groups 2 and 4 are treated with retinoicacid under identical conditions, as described above, to induce the samedegree of apoptosis. Zebrafish embryos belonging to groups 3 and 4 arethen exposed to the test compound. All embryos are then stained with thedye and images are collected using an epifluorescence microscope (NIKONE600) equipped with a CCD camera for low light level detection.Zebrafish embryos from each group are then compared using image andanalysis software. Group 1 embryos serve as (normal) control embryos.Group 2 embryos provide a control for the level of apoptosis induced byretinoic acid. Group 3 embryos demonstrate the ability of the testcompound to induce apoptosis. Group 4 embryos represent the ability ofthe test compound to induce or suppress apoptosis relative to retinoicacid. Absolute changes in the signal area of apoptosis and the number ofapoptotic cells are used to determine if the test compound has had aneffect on apoptotic activity.

Notably, methods of the present invention are performed in live,transparent teleost embryos. The effect of a test compound on cell deathactivity (e.g., apoptotic or necrotic activity) in vivo can bedetermined over time by examining the above-identified zebrafish embryogroups at 24-hour intervals, for a period of up to 4 days. The effect ofa compound on the death of cells of a particular organ (e.g., brain) ortissue of a teleost can be examined over time. Organ-specific andtissue-specific patterns of cell death can be identified. Furthermore,the persistence and duration of the effect of the compound can bedetermined by methods of the invention. In addition, the effect of acompound on either or both the entire whole embryo or specific organsand tissue systems (e.g., the cardiovascular system, the enteric systemand the musculature system) can be determined in vivo simultaneously orindependently. Because teleosts, such as zebrafish, are easy to generateand the assay is readily reproducible, a large number of test compoundscan be easily and quickly screened for their respective effects on andregulation of cell death activity, including apoptosis and/or necrosis.

With methods utilizing benzothiazolium-4-quinolium dye, one cannotdistinguish between a potential effect a particular compound may have onapoptotic processes and necrotic processes. In a developing embryo,necrotic cell death rarely occurs unless the embryo is damaged bynonphysiological injury caused by, for example, physical manipulation.To eliminate nonphysiological injury to an embryo (and thus to eliminateany cell death resulting from necrosis), dechorionated embryos aremaintained in agar coated wells. The agar coating prevents abrasions tothe ectoderm of the embryos. Such abrasions can occur when the embryoscontact plastic surfaces. To prevent further nonphysiological injury toembryos, each embryo is not handled once it is placed into a well of themulti-well culture plate. Staining, compound exposure, and observationson the embryos can all be performed in the multi-well culture platewithout manipulating the embryos, thereby reducing the possibility ofnecrotic damage to the embryos. Specific fluorogenic substrates whichreport enzymatic activity (e.g., caspase enzymatic activity) can be usedin transparent teleost embryo and can aid in distinguishing betweenapoptotic and necrotic activity.

Because teleost embryos, such as zebrafish, can be maintained in smallfluid volumes (e.g., 100 μl) for the first four to five days ofdevelopment, single embryos can be kept in individual wells of amulti-well (e.g., 96-well) culture plate. Alternatively, multipleembryos (e.g., 10 embryos) can be kept in each well of a 24- or 48-wellculture plate, or the like. This makes it possible to detect signals,including, e.g., fluorescent, colorimetric, radioactive andchemiluminescent signals using standard microtiter plate readers and toautomate methods of AP staining and detecting a variety of compounds fortheir effects on cell death activity.

In addition to automating detection, sample handling can be automatedfor rapid reproducible application of dyes and compounds to the teleostsusing methods described herein. To increase the throughput of a compoundapplication, currently available robotic systems (such as the BioRobot9600 from Qiagen, the Zymate from Zymark or the Biomek from BeckmanInstruments)—most of which use the multi-well culture plate format—canbe used with methods of the invention. Well-known and commerciallyavailable instrumentation system can be employed to automate in situhybridization and data recording and retrieval systems and other aspectsof the screening methods of the invention.

The present invention also provides methods of screening a compound foran effect on cell death activity in vivo which comprise administeringthe compound to a teleost in vivo and detecting a response in theteleost indicating an effect on cell death activity, wherein a libraryof compounds is screened for an effect on cell death activity, includingapoptotic and necrotic activity. In some such methods, the library ofcompounds comprises natural compounds. In other such methods of theinvention, the library of compounds comprises synthetic compounds. Inyet other methods of the invention, the library is a combinatoriallibrary. Methods of the invention are useful to screen compound andchemical libraries for molecules which repress or trigger cell death,including repressing or triggering apoptosis or necrosis.

IV. Methods of Screening an Agent for Toxic Activity

A. Whole Animal Toxicity Testing

The predominant methods for toxicity testing use cell-based assays toevaluate the potential impact of different compounds on human and animalhealth. The cytotoxic effect of chemicals on mammalian cells isprimarily measured by cell viability and unscheduled DNA synthesis.Because these toxicity screens are designed to evaluate the in vitroeffect of a compound against cellular targets, they are limited in theirability to predict effects at the organism level, including lethality.In contrast, use of whole animals for toxicity testing addresses thelimitations of cell-based assays and permits simultaneous evaluation atthe molecular and cellular levels.

Whole embryo testing has previously been performed on invertebrates,including fruitfly and nematode (Eisses, Teratog. Carcinog. Mutagen9:315-325 (1989); Hitchcock et al., Arch. Environ. Contam. Toxicol.33:252-260 (1997)). However, because invertebrates are not closelyrelated to humans and lack many of the same organs and enzymes, the useof such results as predictors of toxic effects in humans are limited.Embryo toxicity testing in mammals falls into two categories: 1) cultureassays using either rat or mouse embryos, and 2) in utero assays inwhich compounds are injected into the peritoneum of a pregnant mouse orrat. Although the whole-embryo mouse and rat culture technique is avalidated method for toxicity testing in vertebrates (Chatot et al.,Science 207:1471-1473 (1980); Circurel and Schmid, Xenobiotica18:617-624 (1988)), toxicity testing using this method is complicatedand only a limited number of expensive assays can be performed. Embryosmust be carefully explanted with the visceral yolk sac and ectoplacentalcone intact at 8.5 days of development. Embryo culture time is alsolimited to 48 hours (Bechter et al., Teratol. 44:29A (1991)). Inaddition, due to the complexity of culture conditions, the incidence ofboth false positives and false negatives is high (Guest et al., Can. J.Physiol. Pharmacol. 72(1):57-62 (1994)). The in utero approach avoidsthese issues; however, this approach is complicated by the fact that thecompounds being tested can be metabolized in the liver of the mother.Further, although the in utero approach is useful for examining prenataleffects, it is not helpful in evaluating toxic effects of a compound onpostnatal development. The frog embryo system is another commonly usedmodel for in vitro toxicity testing; however, because frog embryos arenot transparent it is very difficult to examine toxic activities againstparticular tissues and organs over time or simultaneously. A methodwhich permits the screening an agent for toxic activity in multipledifferent organs and tissues of an animal simultaneously and/or in thewhole animal in vivo is needed.

B. Toxic Activity Screening Methods

The present invention provides methods of screening an agent for a toxicactivity in an intact, whole animal and in tissues and organs of wholeanimals in vivo or cells in in vitro using cells of the animal. Suchactivity can be assessed relative to contemporaneous and/or historicalcontrol teleosts (or teleost tissues, organs, or cells) to which theagent has not been administered. Such methods comprise administering theagent to a teleost and detecting a response in the teleost indicatingthe activity. Such methods are useful for rapidly, comprehensively, andreproducibly screening for and predicting toxic responses, includingharmful and lethal effects on developing organs and tissues in wholeteleosts, due to particular agents.

The zebrafish is among the preferred teleosts for these methods. Asoutlined in detail above, zebrafish offer a number of advantages fortoxicity testing, including that zebrafish are transparent (thusfacilitating observation and analysis of multiple tissues and organssimultaneously), develop rapidly, are easy and inexpensive to generateand maintain, and amenable to high throughput toxicity screens. Inaddition, the morphological and molecular bases of tissue and organdevelopment are generally either identical or similar to othervertebrates, including man, and thus toxicity screens of compounds inzebrafish provide relevant information about the effect of compounds inhumans. Moreover, we have determined that teleosts exhibitdose-responsiveness to toxicity and thus zebrafish and the toxicityscreening methods described herein are useful in determining the effectsof particular doses of agents on particular organ and tissue systems andthe sensitivity of particular organs and tissues to such doses.

As described above, the compound to be screened can be administered tothe teleost by diffusion simply adding it to the media containing theteleost or by microinjection or similar techniques which would be knownto one of ordinary skill in the art.

The present invention includes in vivo methods for screening agents fora toxic effect or activity on one or more organs (e.g., the kidney,pancreas, cardiovascular system, central nervous system, liver, etc.) ortissues simultaneously or independently. Also included are in vitromethods in which an agent is administered to a culture of cells of theanimal and the response indicating activity is detected in the cells.All such methods can be used to screen a wide range of agents andcompounds, including, among other things, chemical compounds,pharmaceuticals, therapeutics, environmental and agricultural agents,industrial agents, pollutants, cosmeceuticals, synthetic or naturalcompounds, drugs, organic compounds, lipids, glucocorticoids, peptides,antibiotics, chimeric molecules. sugars, carbohydrates, etc. Theseagents and compounds can be screened singly or as mixtures, includingcomplex mixtures.

The methods of the present invention allow for investigation ofmolecular methods for assessing key liver and kidney enzymes asbiomarkers for organ toxicity. Subtractive library techniques andmultiple enzymatic assays, in combination with drugs of known toxicity,can be used to identify new genes involved in drug response andmetabolic activation phenomena and thus contribute to establishing andvalidating new biomarkers.

Toxic effects and activity resulting from administration of a compoundto an animal (e.g., teleost) can be indicated by a variety of responsesin the animal, including, but not limited to, e.g., molecular changes,genetic mutations, developmental defects, developmental delay,genotoxicity, reproductive toxicity, organ toxicity or dysgenesis,behavioral toxicity, teratogenicity, death of the animal etc.) Responsesindicating toxic activity can be detected in the whole teleost or in atleast one tissue or organ of the teleost. The response can be detectedin multiple tissues and organs simultaneously or separately over time atpredetermined time intervals. For example, the response can be detectedin at least two different tissues, at least two different organs, or inat least one organ and at least one tissue. In in vitro methods, theresponse is detected in one or more cells of the teleost.

Additionally, a response indicating toxic activity can be detected as achange in a gene expression (mRNA) profile for one or more cells,tissues, organs of the animal, or the whole entire animal, by extractingand measuring the level(s) of one or more mRNAs expressed in suchcell(s), tissue(s), organ(s) or the entire teleost at a certain timefollowing agent administration and/or under a specific set ofconditions. To do this, subtractive library experiments can beperformed. mRNA from the control (untreated) and experimental (treated)embryos are extracted at an early and late response period. Thesubtractive libraries are constructed with the polymerase chain reaction(PCR)-Select cDNA Subtraction System (CLONTECH Laboratories, Inc.).Those genes from the embryo that are differentially expressed as aconsequence of the exposure to the compound are selectively isolated,cloned and characterized using standard procedures. The cDNAs are usedto construct a cDNA microarray.

A response indicating toxic activity can also be detected as a change ina protein expression profile for one or more cells, tissues, organs ofthe animal, or the whole entire animal, by extracting and measuring thelevel(s) of one or more different proteins expressed in such cell(s),tissue(s), organ(s), or the entire animal at a certain time followingcompound administration and/or under a particular set of conditions. Inthis protein-based approach, differences in post-translationalmodification or processing such as cleavage can be examined usingtwo-dimensional polyacrylamide gel electrophoresis. Extracts fromcontrol embryos and those exposed to compounds can be directly comparedin the same gel by tagging each extract with a different fluorophoreprior to electrophoretic separation. The tags have no effect on therelative migration of labeled proteins during electrophoresis. Proteinsthat appear unmodified in both samples appear as spots composed of bothfluorescent dyes. Proteins that differ between the two samples, as aresult of cleavage, phosphorylation, etc., fluoresce as tagged in theoriginal extracted sample.

One of ordinary skill in the art would recognize that a variety oftechniques can be used together or separately to generate a signal(e.g., in situ hybridization, antibody staining of specific proteins,etc.) and to detect and assess responses (e.g., colorimetry,fluorescence microscopy, light microscopy, digital image analyzing,standard microplate reader techniques, fluorometry, includingtime-resolved fluorometry, and chemiluminescence, visual inspection, CCDcameras, video cameras, photographic film, or the use of currentinstrumentation such as laser scanning devices, fluorometers,photodiodes, quantum counters, plate readers, epifluorescencemicroscopes, scanning microscopes, confocal microscopes, flowcytometers, capillary electrophoresis detectors, or by means foramplifying the signal such as a photomultiplier tube.

C. Screening Agents for Toxic Activity and/or Angiogenesis Activityand/or Cell Death Activity Simultaneously

The methods for screening agents for toxic activity described herein canbe combined with other methods of the present invention, includingmethods of screening agents for angiogenesis activity (Section II) andcell death activity (Section III). As noted previously, with transparentteleosts, it is possible to assess such multiple activities and theresponses resulting from such activities in the whole teleost or in oneor more tissues or organs simultaneously and at predetermined timeintervals. Assays combining toxicity screening with screening for celldeath activity are useful as discussed previously for identifyingdeleterious and lethal responses resulting from agent administration,proper dosage amounts, and in developing effective therapeutics andtreatment programs.

A variety of techniques can be used together or separately to analyzemultiple activities and responses, including fluorescence microscopy,light microscopy, digital image analyzing, standard microplate readertechniques (colorimetry, fluorometry, including time-resolvedfluorometry, and chemiluminescence), in situ hybridization, antibodystaining of specific proteins, enzymatic changes, changes in proteindistribution temporally and spatially in the teleost, etc.

In one aspect, the present invention provides a method of screening anagent for a toxic activity as described above which further comprisesscreening the agent for an effect on cell death activity by detecting aresponse in the teleost indicating an effect on cell death activity (asdiscussed above). Tissue and organ specific patterns of cell death canbe evaluated in addition to examining various markers to analyze organtoxicity. Cells undergoing cell death can be identified by a variety ofmeans, including those discussed above (e.g., using amembrane-impermeant, nuclear-staining dye from thebenzothiazolium-4-quinolium dye family, the TUNEL assay, or colorimetricor fluorometric enzymatic assay of caspase activity).

In another aspect, the invention provides a method of screening an agentfor a toxic activity as described above which further comprisesscreening the agent for an increase or decrease in angiogenesis bydetecting a response in the teleost indicating an increase or decreasein angiogenesis activity.

Examples

1. Screening Compounds for Toxic Activity on Liver and Kidney inZebrafish

A. Methods

1) Embryo Collection

Wildtype zebrafish embryos were generated by natural pair-wise mating,sorted for viability, and collected as described in Section II forscreening methods for angiogenesis activity. Embryos before being sortedfor viability. Because the fish embryo receives nourishment from anattached yolk sac, no additional maintenance was required.

2) Compound Screening

As discussed above, a variety of compounds can be screened using themethods described for toxic activity on whole animals (e.g., teleosts)and specific organs and tissues. The developmental toxicity effects oftherapeutic/pharmacologic compounds can also be studied; results withsuch compounds using teleosts can be compared with the results obtainedby NCI using mammalian models. By such comparison, the use of themethods and animal models of the invention for predictive assays fordevelopmental toxicity effects of potential therapeutic compounds can beassessed.

3) Maintenance of Embryos and Administration of Compounds

Fertilized zebrafish embryos were obtained by natural spawning in ouraquaculture facility. To reduce variations between batches, randomizedsamples of embryos from 3 or 4 independent matings were used. The testmedium was prepared by combining 5 g of Instant Ocean Salt with 3 g ofCaSO₄ in 25 liters of distilled water, according to Westerfield, supra.The embryos were maintained in the test medium throughout theexperiments. Embryos at 24 hours of development (with chorion) wereexposed continuously for five days to chemical compounds at differentconcentrations of the chemical compounds and controls. In general, theconcentrations ranged from 100 nanomolar (nM) to 100 micromolar (μM).Tests were repeated four times for each series of dilutions, and astandard deviation was calculated for each treatment (see “StatisticalMethods” section). Ten embryos per concentration were exposed in a totalvolume of 1 ml (constant ratio of 100 μl/embryo) using a 24 multi-wellplate. (Other sizes of multi-well plates, such as 96-well plates canalso be used to facilitate screening.) The compounds were renewed daily.In all cases, 0.1% of dimethyl sulfoxide (DMSO) was used as a carriersolvent during the treatment. Controls with and without 0.1% DMSO wereperformed in all experiments. This approach has long been used tointroduce anesthetics and other chemicals into fish embryos(Westerfield, supra).

Experiments were carried out at a constant temperature (28-28.5° C.) inthe dark to protect the compounds from decomposition due to lightexposure. Dead embryos were removed daily, counted, and used tocalculate the Median Lethal Concentration (LC50, see “StatisticalMethods” section herein). Each day, surviving embryos were analyzedvisually under a dissecting binocular microscope (Zeiss, amplification30-50×). Macroscopic malformations (such as axial defects,embryolethality, growth inhibition, general malformations, includingmicrocephaly, macrocephaly, tail truncation, tail malformation, loss ofaxial structures, such as somites, etc.) were observed, classified, andcounted to assess whole animal toxicity. Compounds that were lethal orinduced these or any noted malformations or disruptions duringdevelopment (e.g., during the first 5 days of development) were furtherexamined for toxic effects on organs. The embryonic developmental stagethat is affected by the toxic compound can be determined. Organ toxicitycan be assessed in surviving embryos using in situ hybridization,enzymatic assays, and immunochemistry procedures.

For therapeutic drugs screened for toxic effects, the Median EffectiveConcentration (EC50)(the median concentration needed to caused adesirable effect on a target) can be determined. The Therapeutic Window(TW)(e.g., LC50/EC50) can also be determined; compounds exhibiting ahigh Therapeutic Window, such as 100 or 1,000, are good potential drugcandidates because toxicity at the therapeutic concentration is low.

4) Tissue and Organ Toxicity Testing

a) In Situ Hybridization

To assay specific tissue and organ degeneration, whole mount in situhybridization with RNA probes labeled with digoxigenin (BoehringerMannheim) can be used. Probes which stain early embryonic tissuesinclude MyoD, for the paraxial mesoderm during somitogenesis; brachyury,for the notochord. Probes which specifically stain organs include krx20and pax2 for detection of abnormal development of the caudal midbrainand anterior hindbrain; c-ret for the presumptive brain, spinal cord andexcretory system (developing kidney; nephric duet, and pronephros); andpes for optic tectum, liver primordium, and gut. In situ hybridizationis carried out as follows: embryos are fixed with 4% paraformaldehyde inPBS and hybridized at 65° C. For visual inspection under a microscope,alkaline phosphate-conjugated anti-digoxigenin antibody is used todetect signals following staining with NBT/X-phosphatase (BoehringerMannheim). Toxicity effects on tissue and organ development and function(e.g., liver and kidney), the expression and inducibility of aconstitutive isozyme LMC2 and dioxin-inducible isozyme LM4B ofcytochrome (Cyt.) P-450 in different organs and tissues byimmunohistochemical localization can be analyzed by using methodsdescribed in Buchmann et al., Toxicol. Appl. Pharmacol. 123:160-169(1993).

Automated in situ hybridization image analysis is readily performedusing alkaline phosphatase-conjugated anti-digoxigenin antibody todetect signals after staining with NBT/X-phosphatase.

b) Assessing Toxic Activity in the Liver by Staining

Toxic activity in the liver of the treated whole animal can be assessedvisually by using a rapid colorimetric staining procedure. Thisprocedure is based on the use of a streptavidin (avidin) conjugatedreporter enzyme, such as peroxidase, to detect naturally biotinylatedcarboxylase enzymes in the liver, gut, and digestive tube of wholeanimals, such as zebrafish embryos. These biotinyl-lysine containingenzymes, such as acetyl-CoA carboxylase and other carboxylases, arepredominantly located in the liver and digestive tube. As a result,staining is organ specific (FIG. 11). Quantitative biotinylatedassessment of the liver can be made. By visual detection ofbiotinylation, size and location of the liver can be determined.

Zebrafish embryos (4, 5 or 6 days old) were fixed with 4%paraformaldehyde for 1 hour at room temperature and treated withmethanol 100% overnight at −20° C. The embryos were rehydrated andwashed with PBST. After washing with PBST, the embryos were incubated inblocking solution (3% BSA, 100 mM NaCl in PBST) for 1 hour and treatedwith a bleaching solution (5% Formamide, 0.5×SSC, H₂O₂ 10%) for 20minutes under natural light illumination. After bleaching, the embryoswere incubated for a second time with the same blocking solution for 1hour and incubated with streptavidin conjugated peroxidase (Pierce)(dilution 1:100 in the same blocking solution) with shaking at roomtemperature for 2 hours. The embryos were then washed twenty minutesthree times with PBST and stained for peroxidase with two differentdyes: Diaminobenzidine (DAB) (Pierce) (insoluble) to assess liverstaining and 2,2′-Azino-bis(3-Ethylbenz-thiazoline-6-sulfonicacid)(ABTS)(Sigma) (soluble) to measure a quantitative visual signalusing a colorimetric method. The DAB staining solution used comprised: 1ml of DAB stock solution (5 mg of Diaminobenzidine/ml in PBS, pH 7.4), 9ml of PBS, 10 μl of H₂O₂ (30%). Normally, specific liver staining wasvisualized in 1-5 minutes. Staining for the DAB solution was stopped byseveral washes with water. The ABTS colorimetric method used 10 ml ofABTS solution (10 mg in 33 ml of 0.1 M Citric Acid/OH, pH 4.35) plus 10μl of hydrogen peroxide (30% stock) and was performed for 30 minutes atroom temperature with at least 5 embryos per condition (1 ml of ABTSsolution/5 embryos). The ABTS staining was stopped with 20% SDS/50%N′N-Dimethyl Formamide. The ABTS signal was detected by measuring theabsorbance of the solution at 405 nanometer (nm) using aspectrophotometer. For each condition, four repetitions were performedand the standard deviation “S” calculated as indicated in “StatisticalMethods” section below.

c) Assessing Toxic Activity in the Kidney by Staining

Toxic activity in the kidney (Pronephros) can be assessed visually bycolorimetric staining of the kidney of the treated whole animal (e.g.,zebrafish). Zebrafish pronephros express high levels of the enzymealkaline phosphatase that can be easily assayed using a chromogenic dye.To stain kidneys in zebrafish embryos, embryos were fixed with 2%paraformaldehyde overnight at 4° C. and then treated with methanol 100%for 30 minutes at room temperature. The embryos were rehydrated andequilibrated in NTMT buffer (50 mM MgCl₂, 100 mM NaCl, 100 mM Tris/HCl,pH 9.5, 0.1% Tween 20) at room temperature and then stained with stainsolution (75 mg/ml NBT and 50 mg/ml X-phosphate, equilibrated in thesame buffer). Normally, specific kidney staining was visualized in 10-20minutes (FIG. 14). This staining method was used to assess the toxicactivity of aspirin and dexamethasone on kidney (see below).

d) Assessing Enzymatic Activity

The methodology for assessing enzymatic activity involves in theexposure of the teleost embryos to different compounds at differenttimes (hour to days) with the subsequent in vitro analysis of theirdifferent enzyme activities. The embryos are incubated in a multi-wellplate in the presence or not (i.e., controls) of different compounds.After exposure, they are used to obtained cell lysate preparations. Theenzymes are assayed by the use of colorimetric or fluorometric dyes inend-point or kinetic experiments. The plates are read in an appropriatemicroplate reader. Multienzymatic microarrays can be constructed.

5) Assessing Organ Toxic Activity of Aspirin and Dexamethasone

In feasibility studies, aspirin and dexamethasone were screened fortoxic activity on zebrafish embryos using the methods of the inventiondescribed herein. Aspirin, a general inhibitor of cyclooxygenases(Bosman, Skin Pharmacol. 7:324-334 (1994)), was previously shown toproduce toxicity in a variety of organs in mammal embryos, including thekidney (Ungvary et al., Teratology 27(2):261-69 (1983)). Dexamethasone,an immunosuppressor (Iida et al., Carcinogenesis 19:1191-1202 (1998)),was previously shown to produce liver and gastrointestinal toxicity andto be hepatotoxic in children undergoing cancer treatment (Wolff et al.,Anticancer Res. 18(4B):2895-99 (1998)).

Results are presented in Table 4. As with aspirin, for dexamethasone theLC50 obtained with zebrafish embryos was similar to the valuespreviously described for mice and rats (Table 4). Liver andgastrointestinal toxicity and a dose-response effect were also observed(FIGS. 12A-12B). The quantitative colorimetric endpoint method discussedabove was used to measure the effect of drug treatment on the liver(FIG. 13). Treatment with 100 μM dexamethasone reduced the colorimetricsignal by about 70%, compared with the untreated embryos (control, 0%dexamethasone, FIG. 13). The change in color correlated well with thereduction in size previously observed in the liver using a chromogenicdye (FIG. 12B, bottom), suggesting the reproducibility and accuracy ofthe assay. The method was quantitative (with good confidence limits),rapid, and easy to perform.

The method can be automated using known instrumentation and techniquesautomated toxic screening.

TABLE 4 Five-Day Zebrafish Toxicity Testing Compared with MammalianModels¹ Zebrafish Mammalian models LD₅₀ ² Specific toxicity Specifictoxicity (from Compound tested (mg/liter) observed LD₅₀ (mg/kg) theliterature³) 1. Aspirin 101 teratogen, kidney, 250 (mice, or.)⁴ kidney,ureter, (Clycooxigenase muscle contraction, 200 (rats, or.)cardiovascular, inhibitor) erratic movements 167 (mice, i.p.)⁵craniofacial, musculoskeletal 2. Dexamethasone 324 liver, gastro- 410(mice, i.p.) liver, gastro-intestinal (immunosuppressor) intestinal  54(rats, i.p.) ¹Including mice and rats. ²The LD₅₀ was calculated asindicated in “Statistical Methods.” ³Data was obtained from TOXNET WebSearch and other sources. ⁴or. = orally ⁵i.p. = intraperitoneal

6) Statistical Methods

a) Estimation of LC50

For the concentrations tested with aspirin and dexamethasone in thesestudies, there was no partial lethality and the geometric mean of theparameters “no mortality” (0%) and “mortality” (100%) of the effectconcentration was taken as the LC50 and binomial confidence limits werecalculated according to Stephan, “Methods for Calculating LD50” inAquatic Toxicology and Hazard Evaluation (F. L. Mayce and J. L. Hamelinkeds.) ASTM STP 634, pp. 65-84. Amer. Soc. Testing Materials,Philadelphia, Pa. (1977).

b) Standard Deviation

The colorimetric liver stain method described above was used to obtainqualitative data (i.e., changes in the size, presence, or location ofthe organ) and to study the significance of the variations found in eachtreatment. For each condition, four repetitions were performed and thestatistical value, with its standard deviation, was used to preparegraphics using Microsoft Excel 97 or similar known, standardsoftware/graphics programs.

7) Teratogenic Effects

Information about additional toxic responses/effects indicating toxicactivity of a compound, such as, e.g., growth inhibition andteratogenesis, including microcephality, macrocephality, tailtruncation, tail malformation, can be evaluated by visual inspectionusing a dissecting microscope (Zeiss, amplification 30-50×). Multipletoxic responses and effects can be assessed rapidly and simultaneouslyin transparent teleost embryos.

8) Assessment of Additional Biomarkers

Commercially available antibodies can be used to detect expression andinducibility of different kidney and liver enzymes byimmunohistochemistry. With these biomarkers, the toxicity of drugs andcompounds, including those having known toxicity, can be investigated.The toxic effects of new drugs to be used in the subtractive libraryscreen can also be readily assessed by the methods described herein.Examples of such antibodies include, e.g., anti-Proton Pump H+/K+ ATPase(kidney, Panvera Corporation); anti-LMC2 and anti-dioxin-inducibleisozyme LM4B of cytochrome P-450 (kidney and liver), as previouslydescribed (Buchman et al., Toxicol. Appl. Pharmacol. 123:160-69 (1993));anti-Glutathione S-Transferase (kidney, liver, Panvera Corporation).

2. Identifying Organ Specific Genes Involved in Compound ToxicityResponses Using Subtractive Library Techniques

As a predictive method for drug toxicity, a multi-parametric toxicitytest would be very valuable and useful. Subtractive library experimentsare useful in developing such an approach. Such methodology allows theidentification of genes that are differentially expressed in a targetorgan as a result of chemical/drug exposure. Currently, thegenes/pathways involved in drug/chemical toxicity response anddrug/chemical metabolic activation are difficult to assay primarily dueto the lack of available probes and substrates. Additional informationregarding organ drug toxicity can be obtained by isolating new genes inthe animal (e.g., teleost) using the subtractive library method. Thegenes are cloned, the expression profiles of the genes are evaluated,and their significance as markers for toxicity is compared with datapreviously obtained in mammals.

A. Subtractive Library Techniques

Genes which are differentially expressed in a target organ as a resultof drug/chemical exposure can be identified by using subtractive librarytechniques as follows. Using selected compounds from our LC50 and organtoxicity methods and analyses described above, zebrafish embryos aretreated to induce organ toxicity. At different time points during drugtreatment, the liver and/or kidney (treated and controls) are dissected.This material is used to prepare subtractive libraries to isolate newgenes differentially expressed in treated and control embryos.Subtractive library techniques (e.g., Clontech, Palo Alto, Calif.) areused to selectively isolate genes. The Clontech PCR-Select system usessuppression PCR and requires only one round of subtractive hybridizationto subtract and equalize cDNAs. In addition, the technique requires verylow amounts of poly A+ RNA prepared from two types of tissue undercomparison; normally 0.5-2.0 μg. Recently, this technique was used toisolate several caffeine up-regulated genes from the pre-B cell line1-8, including IGF-1B, and a predicted homologue of the natural killercell antigen, NKR-P1 (Hubank and Schatz, Nucleic Acids Res. 22:5640-5648 (1994)).

The genes identified by the subtractive library technique are selected,and the expression pattern of these genes in the embryos duringcompound/drug exposure can be evaluated. The expression pattern can becompared with the pattern found in mammalian homologues under similarconditions. Genes that serve as “good” indicators or marker of toxicity(e.g., organ toxicity) can be identified and selected.

B. Transgenic Teleosts

Adequate regulatory regions upstream from the target genes withpredictive toxicity value response can be used to construct transgenicteleosts (e.g., zebrafish) carrying reporter genes. The 5′ upstreamregion of these genes is analyzed in order to use the regulatory regionto control the expression of reporter genes in transgenic zebrafish. Inthis approach, genes isolated using subtractive library techniques areused to analyze the 5′ regulatory region. To construct plasmids carryinga reporter gene, such as the Green Fluorescence Protein (GFP) under thecontrol of those regulatory regions, those upstream regulatory regionsthat are adequate in size (1 or 2 kilobases) and expression profile areemployed. These plasmids are used to produce transgenic fish asdescribed in Long et al., Development 124:4105-4111 (1997). For example,because zebrafish are transparent, cells in transgenic zebrafish thatexpress GFP (a reporter gene in specific organs and tissues) can bedetected in vivo using standard fluorescence-based detection techniques;specifically, when cells expressing GFP are illuminated with blue orultraviolet (UV) light, a bright green fluorescence is observed.Light-stimulated GFP fluorescence technique does not require cofactors,substrates or additional gene products and therefore screenings can beperformed in vivo, and using the same embryos, toxicity effects can bemonitored over time using, e.g., a fluorescence plate reader. Using thisscreening method, many genes involved in a drug response which wouldotherwise be difficult to assay can be easily assessed.

C. Zebrafish cDNA Microarrays

cDNA microarray technology can be used to profile complex combinationsof gene expression in drug toxicity response and metabolic activationphenomena. Gene expression of specific organ toxicity can be monitoredusing a microarray of selected zebrafish genes isolated by thesubtractive library techniques discussed above and other sources ofgenes. The cDNA arrays are simple and permit direct readout ofhybridization results; thus, they constitute an ideal technique forstudying gene expression patterns in tissues undergoing drug treatmentat different timepoints (Heller et al., Proc. Natl. Acad. Sci. USA94:2150-2155 (1997)).

V. Screening Automation

The multi-parametric methodology described above can be automated usingstandard instrumentation and computer software programs, permitting thescreening of hundreds of agents per week. These methods combine thephysiological advantages of the teleost and simplicity of agent additionwith the ease of sample handling and detection provided by microtiterplates and associated dispensing and detection apparatus. The methodsare premised, in part, on the observation that teleosts can be culturedand develop normally within the confined space of microtiter wellsnotwithstanding the accumulations of waste products and low availabilityof oxygen due to the confined space. Accordingly, large numbers ofagents can be screened in parallel in the wells of a microtiter platecontaining fertilized embryos. As for other screening methods discussedabove, because teleost embryos develop normally in 100 μl of water,agents and dyes can easily be added to the medium. Furthermore, becausetransparent teleost embryos become opaque when they die, embryolethality is comparatively straightforward to identify using a standardmicrotiter plate reader to calculate the LC50. In addition, a GFPtransgenic fish can be monitored over time in a microplate reader.

An exemplary scheme for performing high throughput screening is shownin. FIG. 15. Zebrafish embryos are contacted with agents to be screenedfor sufficient time for the agent to elicit a response indicative of apharmacological activity in the zebrafish. The amount of time depends onthe assay and can range from 1 hour to 7 days. Different wells can beused to test different agents, and/or to test the same agent atdifferent concentrations. In some methods, each well contains a singlezebrafish and in some methods, each well contains multiple zebrafish.After treatment, wells are analyzed to determine continued viability ofzebrafish (e.g., by determining absorbance at 550 nm). Death ofzebrafish results in high absorbance. Wells containing agents resultingin high lethality are not further pursued, but the identity of suchagents can be stored in a computer file.

Wells containing viable zebrafish are then assayed for pharmacologicalactivity of the agents being tested. This activity is often a change inconcentration of a cellular marker or in the number of cells expressinga cellular marker. This activity can be detected using a substrateprocessed by the enzyme to generate an optically detectable product.After performing the assay, the plates are read for an optical signalindicating pharmacological activity. Agents showing good activity in theassay are then subjected to a confirmatory assay. This assay can beperformed in a microtiter format as before, or can be performed by adifferent format on a teleost (e.g., a microscopic assay), or can beperformed on a higher organism. In some methods, the confirmatory assaydetects the same response as the primary assay but by a different means.For example, the primary assay can detect an increase in enzyme activitycolorimetrically using a microplate reader, and the confirmatory assaycan detect the same enzyme activity on a micrograph. Agents showing goodactivity are also tested for toxicity and lethality. These assays can beperformed in a microtiter format or using other methods described above.Suitable enzymes whose activities are indicative of a toxicity responsein the heart or liver, and substrate for detecting them are described inthe Examples that follow.

The equipment used in the above assays typically includes a multiwellplate, apparatus for dispensing embryos, apparatus for dispensing smallvolumes to the wells, apparatus for mixing fluids in the wells,apparatus for washing the wells, a coding to label wells and the plate,a plate reader for detecting an optical signal from the wells and forreading the code and a computer for controlling the other apparatus andfor storing data. For dispensing teleosts, an automated particlehandling device equipped with a light scattering detection system whichcan contribute about 1 mm particles and distinguish dead embryos (white)from live embryos (transparent) is suitable (BD Biosciences, Bedford,Mass.). To add and remove liquid (e.g., media, washing solutions,labeling reagent solutions), an automatic microplate pippetting andwashing workstation (Biomek, Beckman-Coulter, Zymark, Hopkinton, Mass.;Packard Bioscience, Groningen, The Netherlands) is suitable. The type ofplate reader depends on the nature of the measurement to be made (i.e.,fluorescence, chemiluminescence, colorimetric or radioactive viascintillation counting). Some commercially available plate readers candetect multiple types of signal. Multi-well plates typically have 96wells, but larger or smaller number of wells can be used. In a standardplate, each well has a volume of about 300 μl but larger or smallervolume wells can be used. Typically, zebrafish are cultured in a volumeless than 1 ml, sometimes less than 500 μl, sometimes 50-200 μl, andpreferably about 100 μl. Several laboratory and robotic systems havebeen developed for the purpose of processing microtiter plates. Thesedevices are designed to increase laboratory throughput and many of thesedevices also provide positive sample identification through the use ofanalytical software and barcode labels. The correlation between identityof agents and wells can be referred to as a correspondence regime.Examples of suitable equipment are described below.

The assays are typically performed on whole teleosts. The teleosts canbe natural or transgenic. The teleosts are living when contacted withagents. In some methods, teleosts are killed before detecting signal. Insuch methods, the agent being tested is typically removed. Teleosts canthen be fixed or lysed to facilitate detection of signal. In somemethods, the teleosts remain living throughout the assay includingdetection of optical activity (such as GFP). In such methods, a seriesof measurements of signal can be made over time on the same teleosts. Insuch methods, the agent being tested and the labeling reagent used totest response can be left in contact with the teleost throughout theassay. In some methods, in which an optical signal is generated within ateleost, the teleost should be sufficiently transparent that the opticalsignal can be detected in the whole teleosts. In other methods, thesignal diffuses out of the teleost, or can be induced to do so bytreatment with lysing agents. In such methods, transparency of theteleost is not necessary.

The extent or rate of appearance of a signal depends on the response ofa teleost to an agent, and the response is in turn an indicator of apharmacological activity of the agent. For example, modulation ofangiogenesis is a pharmacological activity, an increase or decrease inalkaline phosphatase activity is a cellular response indicative ofangiogenesis, and an OD405 reading due to processing of the alkalinephosphatase substrate pNPP is an optical signal that depends on thecellular response. Similarly, modulation of apoptosis is apharmacological activity, an increase or decrease in caspases activityis a response indicative of modulation of apoptosis and varioussubstrates of caspases are suitable labeling reagents for generating anoptical signal dependent on the response.

In general, a pharmacological activity is a property of an agent thatindicates that the agent is, or may be, useful for treatment and/orprevention of a disease. Thus, pharmacological activity includesprophylactic and therapeutic activities as defined herein. In general,responses indicative of pharmacological activity can include: 1) aincrease or decrease in the number of cells or the concentration acellular marker, such as an enzyme or secondary metabolite in a cell, 2)the modulation of a cellular pathway, for example, by binding of anagent to a cellular receptor, or 3) the promotion or inhibition of aphysiological event such as cell growth or differentiation. Examples ofcellular markers that can be detected include nucleic acids and proteinsand enzymes. Nucleic acids can be detected by a hybridization assayusing a probe nucleic acid. Usually, the probe nucleic acid is labeledalthough secondary labeling schemes are also possible. Proteins can bedetected using antibodies that specifically bind to the proteins. Insome methods, the antibody is directly labeled and in other methods asecondary labeling scheme is used. Enzymes can be detected using asubstrate processed by the enzyme to generate an optically detectableproduct. Modulation of cellular proliferation can be determined fromcorresponding modulation of component molecules, particularlyconstitutive expressed proteins, or nucleic acids.

Optical signals useful for monitoring cellular response include color,fluorescence, chemiluminescence, opacity, and radioactivity, the lattercan be detected via scintillant induced light scattering. Responses canbe detected by light scattering or photon counting or other methods. Insome methods, a product having an optical signal is generated by areaction within cells of the teleosts, and is an indication of the levelof a particular enzyme catalyzing the reaction. In other methods, thelabeling reagent is attached directly to an agent, such as antibody,that binds to a cellular molecule. In some methods, the labeling reagentis attached to a secondary-labeling reagent that binds to a primaryreagent, which in turn binds to a cellular molecule.

Usually some wells of the multiwell plate are occupied by positive andnegative controls. Positive controls can be agents (s) known to have thepharmacological activity being tested for and negative controls,agent(s) known to lack the pharmacological activity. In some methods,multiple positive and/or negative controls are distributed at differentlocations on the plate.

Microplate assays can also be used to monitor absorbance, excretion,metabolism or intracellular distribution of a agent in teleosts. In suchmethods, the wells provide a means to contain teleosts while a agentredistributes between media and the teleosts, and/or is metabolizedwithin the teleost. Initially, the agent can be in the medium only, inthe teleost only, or in both the teleost and the medium. After culturingthe teleost for a period, the amount of the agent in the medium, or theteleost or both is determined. A decrease in the amount of agent in themedium over the incubation period is a measure of absorption of theagent and allows calculation of an absorption rate. An increase in theamount of agent in the medium over the incubation period is a measure ofexcretion of the agent and allows calculation of an excretion rate. Anincrease in amount of agent in the teleosts over the incubation periodreflects a net absorption. A decrease in amount of agent in the teleostover the incubation period reflects a net excretion. By performing theassay with different initial concentrations of agent in the media andthe teleosts, it is possible to calculate the rates of both of theseprocesses. In methods in which the detection assay distinguishes betweenthe agent and metabolic products of the agent, it is also possible tocalculate a metabolism rate.

In some methods, the agent itself generates an optically detectablesignal or can be labeled with a secondary labeling reagent that givesrise to such a signal. The combined amount of agent in medium andteleost is detected with microplate reader. After detection, the mediumis discarded and the amount of agent within the teleosts is detected.

In some methods, when the agent is not labeled, the amount of agent inmedia can be determined by extracting the medium with chloroform (mostsmall molecule agents are soluble in chloroform) or other organicsolvent to isolate the agent, and quantifying the agent by HPLC/MSdesigned for automation. The agent within teleosts can be extracted withan organic solvent such as chloroform. The extracted agent and itsmetabolite can be analyzed by HPLC/MS designed for an automation system.In some methods, the teleost is fixed after culturing with the agent,and the location of agent within the teleost is determined bymicroscopic examination.

Examples

A. Procedure for High Throughput Screening

1. Synchronized embryos are distributed into 96-well assay plate.Embryos developmental stage (age) can be synchronized by treating theadult fish with propidium iodide, and stimulating them to lay egg in apredicted time frame. Embryos can be distributed using an automatedparticle handling devices equipped with a light scattering detectionsystem (BD Biosciences, Bedford, Mass.). The particle handling deviceshould preferably be capable of dispensing up to 600 μm particles.Alternatively a liquid dispenser fitted with large bore pipette tip canbe used to dispense embryos into microtiter wells automatically.

2. 100 μl of diluted compounds are added to each well (in fish water).An automatic microplate pippetting and washing workstation (Zymark,Hopkinton, Mass.; Packard Bioscience, Groningen, The Netherlands) can beused. A simple bar code system to identify each plate (Decitek,Westborough, Mass.).

3. Plates are incubated at 28° C. for designated time period in anincubator.

4. Plates are assessed for dead embryos. Such assessment can beperformed by visual inspection or by quantitative determination ofabsorbance at 550 nm. Dead embryos become opaque and disintegrate, andcause the clear culture solution to become turbid. This turbid solutionusually absorbs light at 550 nm. Absorbances at 550 nm exceeding athreshold are scored as dead embryos, and this information is recordedin a lethal compound data base using the bar code system to read theidentity of compounds causing lethality. Wells containing lethalcompounds are eliminated from subsequent analysis steps.

5. The plate is washed to remove residual compound and fish water. Inthis and other wash steps, a vacuum manifold with adjustable vacuumpressure to remove the washing solution, and prevent the embryo tissueloss.

6. 100 μl ice cold 70% EtOH is added to each well.

7. The 70% EtOH is removed.

8. 100 μl of 100% EtOH is added.

9. The wells are incubated at −20° C. for 30 minutes in a −20° C.freezer.

10. The 100% EtOH is removed.

11. 100 μl of appropriate buffer is added. The mixture is incubated for10 min at room temperature. The procedure is then repeated 2 more times.

12. The above buffer is removed.

13. 100 μl substrate solution for an enzyme assay is added. The mixtureis incubated at room temperature for designated time period

14. 100 μl of stopping solution is added.

15. A colored metabolic product resulting from an enzymic activity beingmeasured is read at the appropriate wavelength in an automaticmicroplate reader (Biotek, Inc. Winooski, Vt.)

B. Assays for Pharmacological Activity

1. Detecting Angiogenesis

Angiogenesis was monitored by quantitative analysis of endogenousalkaline phosphatase (EAP) levels using the following procedure.

a. Embryos were dehydrated through 70% EtOH, then 100% EtOH

b. Embryos were incubated in 100% EtOH

c. Embryos were re-hydrated and equilibrated by 1M diethanolamine bufferpH 9.8

d. 100 μl of 0.5 mg/ml p-nitrophenyl phosphate (pNPP kit #37620, Pierce)was added as substrate

e. incubation time was determined by testing (see following results)

f. 50 μl of 2N NaOH was added to stop the enzyme reaction

g. OD at 405 nm was recorded by a ELISA plate reader

We used normal zebrafish embryos at 24, 48 and 72 hours postfertilization with known patterns of vessel development as controls toestablished the optimal conditions for distinguishing different stagesof vessel development quantitatively. Since the enzyme substrate (pNPP)comes in a fixed concentration, we defined the optimal incubation timefor assessing different quantities of the enzyme. FIG. 16A shows atypical enzymatic kinetic curve between the OD reading and the substrateincubation time. A linear relationship can be established between the 20and 40 minute time points. Based on this same data, the OD reading vs.embryo age for each incubation time were replotted to determine theoptimal incubation time (FIG. 16B). 30 minutes is the optimal incubationtime for distinguishing the difference in EAP level for embryos at 24,48 and 72 hpf.

To demonstrate a correlation between results using the quantitativeenzyme assay and microscopic observation of vessel development, aseparate set of experiments was performed. Embryos, processed asdescribed above, were measured directly for OD (405 nm) after incubatingwith pNPP for 30 minutes without adding the stopping reagent (2N NaOH).The OD results were similar to those with stopping agent added. Thesoluble substrate pNPP was washed off afterwards and the embryos werere-equilibrated into NTMT buffer and re-stained, as described in vesselstaining for microscopic examination. The image is shown in FIG. 17.This result shows that the enzyme assay detects differences in vesseldevelopment, similar to results using microscopy.

The enzyme assay was then used to assess the effect of differentconcentrations of SU5416 on the embryos. Embryos with intact chorion at24 hpf were collected and distributed into a 96-well plate. SU5416 at 0,0.25, 0.5, 0.75, 1.0 and 1.5 μM (0.1% DMSO as carrier) was addeddirectly into fish water and embryos were incubated continuously for 2days at 28° C. At the end of the incubation, SU5416 was washed off usingfish water, and embryos were dehydrated into EtOH and processed, aspreviously described. 24 hpf (day 1), embryos without chorion were usedas the baseline (0%), and 72 hpf (day 3) normal embryos were used as100% control.

The plot shown in FIG. 18 demonstrates a clear dose response curve; 50%of vessel development is inhibited with a 1.6 μM concentration ofSU5416. It also indicates that the agent carrier (0.1% DMSO) does notcause a significant effect on vessel development. The OD measurement ofthe vessel development in these SU5416 treated embryos was alsocorrelated by microscopy. Although there was a direct correlation, asexpected, the quantitative enzyme assay was more sensitive.

2. Detecting Liver/Gut Toxicity

Several carboxylase enzymes containing biotin covalently bound as thecoenzyme are present in the liver and gut of zebrafish embryos(Gregolin, et al, 1968).] The specific expression of these enzymes inthe liver and gut in both control and agent treated embryos was observedby binding to avidin detected by microscopy. These parameters were thenused to develop an ELISA-type assay to measure the level of carboxylase,which reflects liver and gut formation/function. The process involvesthe following steps.

(1) embryos are dehydrated through 70% EtOH, then 100% EtOH

(2) embryos are incubated in 100% EtOH

(3) endogenous peroxidase activity is suppressed by adding 25 μl ofperoxidase suppressor (Prod. # 35000, Pierce) for 30 minutes

(4) Embryos are washed with PBS, pH 7.4 three times, 10 minutes each

(5) Embryos are incubated with 100 μl of Avidin-Biotin-horseradishperoxidase (A-B-HRP) (Kit # 32050, Pierce) for 1 hour

(6) Excess A-B-HRP is washed off with PBS

(7) Equilibrate embryos into 1M citrate buffer pH 5.0

(8) The embryos are incubated with 100 μl HRP substrate3,3′,5,5′-tetramethylbenzidine (TMB, kit # 34021, Pierce) at varioustime interval to determine the optimal incubation time

(9) 100 μl of 1M H2SO4 is added to stop the reaction

(10) OD at 450 nm is recorded to determine the carboxylase level

The linear relationship between the OD (450 nm) reading and incubationtime is established, and the optimal substrate incubation timedetermined (at the mid-point in the linear range). OD (450 nm) isplotted against embryo stage. The slope between two points defines thedifference in OD reading (ΔOD) between embryos at different stages. Thesharpest slope gives the greatest difference in OD measurement, which ismost useful in distinguishing the difference in carboxylase level, andreflects the difference in organ development. This parameter is used todetermine the optimal embryonic stage for agent testing.

Correlation of quantitative measurement and qualitative observation ofliver/gut is also performed based on the principle described above.Embryos 24 hours post fertilization (hpf) to 144 hpf are assayed at thedetermined optimal substrate incubation time as above, except, insteadof adding 1M H2SO4 to stop the enzymatic reaction, the supernatant isremoved from each well and transferred to a new plate to be measured forOD at either 370 nm or 652 nm. The embryos are washed in PBST (PBS plus0.1% Tween 20) to remove residual TMB substrate and re-equilibrated forDBA staining. The DBA staining solution is prepared following themanufacturer's recommended protocol (1 ml of 0.5% DBA stock solution, 9ml of PBS, 10 μl of 30% H₂O₂). Specific liver & gut staining isvisualized in 1-5 minutes. Staining is stopped by several washes withwater. Embryos are visualized on a dissecting microscope (Zeiss Stemi2000-C). The size of the visualized image of liver/gut is compared to ODmeasurements.

Additional enzymes and their substrates that can be used to assess liverfunction in this format include:

TABLE 5 Enzyme Substrate Source Cytochrome P450 1A1 methoxyresorufinSigma Superoxide Dismutase 5,6,6a,11b-tetrahydro-3,9,10 Calbiochem Kittrihydoxybenzo[c]fluorene Glutathione-S-transferase P1-15-((4′-(S-Glutathionyl))-2′,3′ Molecular Probes, 5′,6′-tetrafluorobenzoylamino) fluoresein Glutathione peroxidase GlutathioneProxidase Assay kit Calbiochem Kit

3. Detecting Heart Toxicity

Heart toxicity can be determined using specific heart proteins,tropomyosin and cardiac myosin heavy chain, as markers. Antibodiesagainst these proteins are used as ligands for these markers. Theantibodies are conjugated with horse radish perodixase (HRP), which isquantified by incubating with the substrate TMB. The steps are similarto those described in liver/gut toxicity assay, embryos are blocked by0.2% bovine serum albumin (BSA) in PBS, then incubated with primaryantibody against the specific protein for 2 hours at room temperature,washed in PBST to remove unbound primary antibody, secondary antibodyconjugated with HRP is added instead of A-B-HRP.

4. Detecting Apoptosis

In response to a variety of apoptotic signaling cascades, caspases,which are normally present in the cell as pro-enzymes (zymogens), areeither cleaved or autocatalytically activated by intracellularsequestration. Caspase activity plays a pivotal role in the initiationand execution phases of apoptosis. The presence of caspase activity canbe monitored in the embryonic zebrafish using commercially availablefluorogenic and colorimeteric caspase substrates specifically engineeredto penetrate the cell.

All reagents (fluorescent stains, chemical agents, and ‘pretreatment’apoptosis inducers for anti-apoptotic screen) are added directly to thefish water in 96 well plates. DMSO (0.2% unless otherwise indicated) wasused as a vehicle or carrier. Controls 1) with tested agent alone, 2)without tested agent, but equivalent amount of carrier plus dye, and 3)no treatment, are usually carried out in parallel.

After brief period of recuperation, the embryos are analyzed with amicroplate reader.

TABLE 6 Enzyme Substrate Source Caspase 1 AC-YVAD-AMC CalbiochemAC-YVAD-pNA Calbiochem Caspase 3 AC-DEVD-AMC Calbiochem AC-DEVD-pNACalbiochem Caspase 2 Z-VDVAD-AFC Calbiochem Caspase 4 AC-LEVD-AFCCalbiochem Caspase 5 AC-WHED-AFC Calbiochem Caspase 6 AC-VEID-AMCCalbiochem AC-VEID-pNA Calbiochem Caspase 8 Z-IETD-AFC CalbiochemAC-IETD-pNA Calbiochem Caspase 9 Ac-LEHD-AFC Calbiochem

5. Detecting Change in Cellular Receptor Expression

The processes described above can be modified by adding a embryo lysingstep after the agent incubation to improve the accessibility ofreceptors to ligands. Ligands for the specific cellular receptors can beeither labeled by fluorescent dye or conjugated with horse radishperoxidase and assessed by the enzymatic reaction.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. The above examples are provided to illustrate the invention,but not to limit its scope; other variants of the invention will bereadily apparent to those of ordinary skill in the and are encompassedby the claims of the invention. The scope of the invention should,therefore, be determined not with reference to the above description,but instead should be determined with reference to the appended claimsalong with their full scope of equivalents. All publications,references, and patent documents cited in this application areincorporated herein by reference in their entirety for all purposes tothe same extent as if each individual publication or patent documentwere so individually denoted.

1. A method of screening a test agent for a toxic activity affectingcardiovascular function in a mammal, the method comprising: (a)contacting a zebrafish with a test agent in vivo, wherein the test agentis administered to the zebrafish though culture media; (b) evaluating aparameter of cardiovascular function in the zebrafish contacted with thetest agent relative to the parameter in a control zebrafish that has notbeen contacted with the test agent to determine whether the parameter isresponsive to the test agent, a difference in the parameter beingindicative of a toxic activity affecting cardiovascular function in thezebrafish contacted with the test agent; and (c) correlating the toxicactivity of the test agent affecting cardiovascular function in thezebrafish with a predicted effect on cardiovascular function in amammal.
 2. The method of claim 1, wherein the parameter ofcardiovascular function is heart rate.
 3. The method of claim 1, whereinthe zebrafish is a wild-type zebrafish.
 4. The method of claim 1,wherein the zebrafish is a zebrafish larva.
 5. The method of claim 1,wherein the method is performed in a multi-well format.
 6. The method ofclaim 1, further comprising contacting the zebrafish with a dye prior tothe step of evaluating the parameter of cardiovascular function, the dyebeing useful for indicating whether the parameter is responsive to thetest agent.
 7. The method of claim 1, wherein the test agent is a smallmolecule.
 8. The method of claim 1, wherein the test agent is a protein.9. The method of claim 1, wherein a plurality of test agents areevaluated.
 10. The method of claim 9, wherein the parameter ofcardiovascular function is heart rate.
 11. The method of claim 9,wherein one of the plurality of test agents is a hormone.
 12. The methodof claim 9, wherein one of the plurality of test agents is a smallmolecule.
 13. The method of claim 1, wherein step (a) comprisescontacting zebrafish larvae with the test agent in the wells of amicrotitre plate.
 14. The method of claim 13, wherein different wells ofthe microtitre plate contain different test agents.
 15. The method ofclaim 13, wherein the test agent in at least one individual well differsfrom the test agent in a second individual well by concentration ordosage.
 16. The method of claim 1, wherein the parameter ofcardiovascular function is heart contractility.
 17. The method of claim16, wherein correlating the toxic activity affecting cardiovascularfunction of the test agent in the zebrafish with a predicted effect oncardiovascular function in a mammal includes identifying a test agentthat causes a change in heart contractility in the zebrafish as acardiotoxic agent in a mammal.
 18. The method of claim 14, wherein thedifferent test agents comprise different small molecules or proteins.19. The method of claim 18, wherein the different compounds aredifferent small molecules.
 20. The method of claim 1, wherein thecardiovascular function is cardiac function.
 21. The method of claim 1,wherein the test agent is added to culture media already containing thezebrafish.
 22. The method of claim 1, wherein the zebrafish has achorion when contacted with the test agent.